Methods and compositions for increasing alpha-L-iduronidase activity in the CNS

ABSTRACT

Provided herein are methods and compositions for treating a subject suffering from a deficiency in α-L-Iduronidase in the CNS. The methods include systemic administration of a bifunctional fusion antibody comprising an antibody to a human insulin receptor and an α-L-Iduronidase. A therapeutically effective systemic dose is based on the specific CNS uptake characteristics of human insulin receptor antibody-α-L-Iduronidase fusion antibodies as described herein.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/397,649, filed Jan. 3, 2017, and issued as U.S. Pat. No. 10,202,467on Feb. 12, 2019, which is a continuation of U.S. patent applicationSer. No. 14/606,239, filed Jan. 27, 2015, and issued as U.S. Pat. No.9,567,400 on Feb. 14, 2017, which is a continuation of U.S. applicationSer. No. 12/179,806, filed Jul. 25, 2008 and issued as U.S. Pat. No.8,974,791 on Mar. 10, 2015, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/952,547, filed Jul. 27, 2007, the contents ofwhich are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 9, 2015, isnamed 28570-704-303-sequence-listing.txt and is 22,760 bytes in size.

BACKGROUND OF THE INVENTION

Type I mucopolysaccharidosis (MPS), also known as Hurler's syndrome, isan inherited metabolic disease caused by a defect in the enzymeα-L-iduronidase (IDUA), which functions to degrade mucopolysaccharides.An insufficient level of IDUA causes a pathological buildup of heparansulfate and dermatan sulfate in, e.g., the heart, liver, and centralnervous system. Symptoms including neurodegeneration and mentalretardation appear during childhood and early death can occur due toorgan damage. Typically, treatment includes intravenous enzymereplacement therapy with recombinant IDUA. However, systemicallyadministered recombinant IDUA does not cross the blood brain barrier(BBB), and therefore has little impact on the effects of the disease inthe central nervous system (CNS).

SUMMARY OF THE INVENTION

Described herein are methods and compositions for treating a subjectsuffering from an IDUA deficiency. In particular, the methods allowdelivery of IDUA to the CNS by systemically administering atherapeutically effective amount of a bifunctional human insulinreceptor antibody-IDUA (HIR Ab-IDUA) fusion antibody. The HIR Ab-IDUAfusion antibody binds to the extracellular domain of the insulinreceptor and is transported across the blood brain barrier into the CNS,while retaining IDUA activity. A therapeutically effective systemic doseof a HIR Ab-IDUA fusion antibody for systemic administration will bebased, in part, on the specific CNS uptake characteristics of the fusionantibody from peripheral blood as described herein.

Accordingly, in one aspect provided herein is a method for treating anα-L-iduronidase deficiency in the central nervous system of a subject inneed thereof, comprising systemically administering to the subject atherapeutically effective dose of a fusion antibody havingα-L-iduronidase activity. The method is characterized by the following:(i) at least about 0.5% of the therapeutically effective dose isdelivered to the brain; (ii) the fusion antibody: comprises: (a) afusion protein containing the amino acid sequence of an immunoglobulinheavy chain and an α-L-iduronidase, and (b) an immunoglobulin lightchain; (iii) the fusion antibody binds to an extracellular domain of thehuman insulin receptor; and catalyzes hydrolysis of unsulfatedalpha-L-iduronosidic linkages in dermatan sulfate; and (iv) the aminoacid sequence of the α L iduronidase is covalently linked to the carboxyterminus of the amino acid sequence of the immunoglobulin heavy chain.

In some embodiments at least about 50,000 units of α-L-iduronidaseactivity are delivered to the brain. In some embodiments, thetherapeutically effective dose of the fusion antibody comprises at leastabout 1×10⁶ units of α-L-iduronidase activity or at least about 140,000units/Kg of body weight. In some embodiments the IDUA specific activityof the fusion antibody is at least 200,000 units/mg. In someembodiments, systemic administration is parenteral, intravenous,subcutaneous, intra-muscular, trans-nasal, intra-arterial, transdermal,or respiratory. In some embodiments, delivery of at least 0.5% of thetherapeutically effective dose to the brain occurs within two hours orless after the systemic administration.

In some embodiments, the fusion antibody is a chimeric antibody.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1 with up to 4 single amino acid mutations, a CDR2corresponding to the amino acid sequence of SEQ ID NO:2 with up to 6single amino acid mutations, or a CDR3 corresponding to the amino acidsequence of SEQ ID NO:3 with up to 3 single amino acid mutations,wherein the single amino acid mutations are substitutions, deletions, orinsertions.

In other embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1 with up to 3 single amino acid mutations, a CDR2corresponding to the amino acid sequence of SEQ ID NO:2 with up to 6single amino acid mutations, and a CDR3 corresponding to the amino acidsequence of SEQ ID NO:3 with up to 3 single amino acid mutations.

In other embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:2, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3.

In further embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:2, and a CDR3 corresponding to the amino acid sequence of SEQ IDNO:3.

In some embodiments, the immunoglobulin light chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:4 with up to 3 single amino acid mutations, a CDR2corresponding to the amino acid sequence of SEQ ID NO:5 with up to 5single amino acid mutations, or a CDR3 corresponding to the amino acidsequence of SEQ ID NO:6 with up to 5 single amino acid mutations,wherein the single amino acid mutations are substitutions, deletions, orinsertions.

In other embodiments, the immunoglobulin light chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:4 with up to 3 single amino acid mutations, a CDR2corresponding to the amino acid sequence of SEQ ID NO:5 with up to 5single amino acid mutations, and a CDR3 corresponding to the amino acidsequence of SEQ ID NO:6 with up to 5 single amino acid mutations.

In other embodiments, the immunoglobulin light chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:5, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6.

In further embodiments, the immunoglobulin light chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:5, and a CDR3 corresponding to the amino acid sequence of SEQ IDNO:6.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:2, and a CDR3 corresponding to the amino acid sequence of SEQ IDNO:3; and the immunoglobulin light chain comprises a CDR1 correspondingto the amino acid sequence of SEQ ID NO:4, a CDR2 corresponding to theamino acid sequence of SEQ ID NO:5, and a CDR3 corresponding to theamino acid sequence of SEQ ID NO:6.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody is at least 90% identical to SEQ ID NO:7 and the amino acidsequence of the light chain immunoglobulin is at least 90% identical toSEQ ID NO:8.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody comprises SEQ ID NO:7 and the amino acid sequence of the lightchain immunoglobulin comprises SEQ ID NO:8

In yet further embodiments, the α-L-iduronidase comprises an amino acidsequence at least 90% (e.g., 95%, or 100%) identical to SEQ ID NO:9.

In other embodiments, the amino acid sequence of the immunoglobulinheavy chain of the fusion antibody at least 90% identical to SEQ IDNO:7; the amino acid sequence of the light chain immunoglobulin is atleast 90% identical to SEQ ID NO:8; and the amino acid sequence of theα-L-iduronidase is at least 95% identical to SEQ ID NO:9 or comprisesSEQ ID NO:9.

In still other embodiments, the amino acid sequence of theimmunoglobulin heavy chain of the fusion antibody comprises SEQ ID NO:8,the amino acid sequence of the immunoglobulin light chain comprises SEQID NO:8, and the amino acid sequence of the IDUA comprises SEQ ID NO:9

In a further aspect provided herein is a method for treating anα-L-iduronidase deficiency in the central nervous system of a subject inneed thereof, comprising systemically administering to the subject atherapeutically effective dose of a fusion antibody havingα-L-iduronidase activity, where the method is characterized in that (i)at least about 0.5% of the systemically administered therapeuticallyeffective dose is delivered to the brain; (ii) the fusion antibody:comprises: (a) a fusion protein at least 95% identical to SEQ ID NO:10,and (b) an immunoglobulin light chain; and (iii) the fusion antibodybinds to an extracellular domain of the human insulin receptor; andcatalyzes hydrolysis of unsulfated alpha-L-iduronosidic linkages indermatan sulfate.

In yet another aspect provided herein is a method for treating anα-L-iduronidase deficiency in the central nervous system of a subject inneed thereof, comprising systemically administering to the subject atherapeutically effective dose of a fusion antibody havingα-L-iduronidase activity, where the method is characterized in that: (i)at least about 0.5% of the therapeutically effective dose is deliveredto the brain; (ii) the fusion antibody: comprises a fusion proteincontaining the amino acid sequence of an immunoglobulin heavy chain andan α-L-iduronidase; or comprises a fusion protein containing the aminoacid sequence of an immunoglobulin light chain and an α-L-iduronidase;binds to the extracellular domain of the human insulin receptor; andcatalyzes hydrolysis of unsulfated alpha-L-iduronosidic linkages indermatan sulfate; and (iii) the amino acid sequence of theα-L-iduronidase is covalently linked to the carboxy terminus of theamino acid sequence of the immunoglobulin heavy chain or theimmunoglobulin light chain.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings, as follow:

FIG. 1. Amino acid sequence of an immunoglobulin heavy chain variableregion from an exemplary human insulin receptor antibody directedagainst the extracellular domain of the human insulin receptor. Theunderlined sequences are a signal peptide, CDR1, CDR2, and CDR3,respectively. The heavy chain constant region, taken from human IgG1, isshown in italics.

FIG. 2. Amino acid sequence of an immunoglobulin light chain variableregion from an exemplary human insulin receptor antibody directedagainst the extracellular domain of the human insulin receptor. Theunderlined sequences are a signal peptide, CDR1, CDR2, and CDR3,respectively. The constant region, derived from human kappa light chain,is shown in italics.

FIG. 3. A table showing the CDR1, CDR2, and CDR3 amino acid sequencesfrom a heavy and light chain of an exemplary human insulin receptorantibody directed against the extracellular domain of the human insulinreceptor.

FIG. 4. Amino acid sequence of human α-L-iduronidase (IDUA) (GenBankNP_000194), not including the initial 26 amino acid signal peptide(mature IDUA).

FIG. 5. Amino acid sequence of a fusion of an exemplary human insulinreceptor antibody heavy chain to mature human IDUA. The underlinedsequences are, in order, an IgG signal peptide, CDR1, CDR2, CDR3, and apeptide linker linking the carboxy terminus of the heavy chain to theamino terminus of the IDUA. Sequence in italic corresponds to the heavychain constant region, taken from human IgG1. The sequence in boldcorresponds to human IDUA.

FIG. 6. An exemplary HIR Ab-IDUA fusion antibody is formed by fusion ofthe amino terminus of the mature IDUA to the carboxyl terminus of theCH3 region of the heavy chain of the HIR Ab. The fusion protein is abi-functional molecule: the fusion protein binds the HIR, at the BBB, tomediate transport into the brain, and expresses IDUA enzyme activity,which is deficient in MPS Type I (Hurler's syndrome).

FIG. 7. Schematic depiction of a “molecular trojan horse” strategy inwhich the fusion antibody comprises an antibody to the extracellulardomain of the human insulin receptor, which acts as a molecular Trojanhorse (TH), and IDUA, a lysosomal enzyme (E). By itself, the IDUAnormally does not cross the blood-brain barrier (BBB). However,following fusion of the IDUA to the TH, the enzyme is able to cross theBBB, and the brain cell membrane, by trafficking on the IR, which isexpressed at both membranes in the brain.

FIG. 8. Ethidium bromide stain of agarose gel of human IDUA cDNA (lane1), which was produced by PCR from human liver cDNA, and IDUA-specificODN primers (Table I). Lanes 2 and 3: PhiX174 HaeIII digested DNAstandard, and Lambda HindIII digested DNA standard.

FIG. 9. Western blot with either anti-human (h) IgG primary antibody(right panel) or rabbit anti-human IDUA primary antiserum (left panel).The immunoreactivity of the HIR Ab-IDUA fusion antibody is compared tothe HIR Ab alone. Both the HIR Ab-IDUA fusion antibody and the HIR Abhave identical light chains on the anti-hIgG Western. The HIR Ab-IDUAfusion heavy chain reacts with both the anti-hIgG and the anti-humanIDUA antibody, whereas the HIR Ab heavy chain only reacts with theanti-hIgG antibody. The size of the HIR Ab-IDUA fusion heavy chain, 130kDa, is about 80 kDa larger than the size of the heavy chain of the HIRAb, owing to the fusion of the 80 kDa IDUA to the 50 kDa HIR Ab heavychain.

FIG. 10. Binding of either the chimeric HIR Ab or the HIR Ab-IDUA fusionprotein to the HIR extracellular domain (ECD) is saturable. The ED₅₀ ofHIR Ab-IDUA binding to the HIR ECD is comparable to the ED₅₀ of thebinding of the chimeric HIR Ab.

FIG. 11. (A) Intracellular IDUA enzyme activity is increased in Hurlerfibroblasts in proportion to the concentration of medium HIR Ab-IDUAfusion protein. Data are mean±SE (n=3 dishes/point). The horizontal baris the IDUA enzyme activity in healthy human fibroblasts (284±5 units/mgprotein). (B) Reversal of glycosaminoglycan (GAG) accumulation in Hurlerfibroblasts with a single treatment of 0.3 μg/mL of HIR Ab-IDUA fusionprotein in the medium. There is a 70% reduction in GAG accumulation, ascompared to the ³⁵S incorporation in healthy human fibroblasts(p<0.0005). Data are mean±SE (n=5 dishes/point).

FIG. 12. (A, B, C, D) Hurler fibroblasts were incubated with HIR Ab-IDUAfusion protein for 24 hours and then fixed and immune stained forconfocal microscopy. The fixed cells were stained with a rabbitpolyclonal antibody to human IDUA (panel A: red channel signal, shownhere in black and white), and a mouse monoclonal antibody to humanlysosomal associated membrane protein (LAMP)-1 (panel B: green channelsignal, shown here in black and white). The overlap image in panel Cshows sequestration of the HIR Ab-IDUA fusion protein within lysosomes.Panel D is an overlap image of negative control primary antibodies:rabbit serum and mouse IgG. (E) Film autoradiography of Rhesus monkeybrain removed 2 hours after an intravenous administration of [¹²⁵I]-HIRAb-IDUA fusion protein. Coronal sections through the forebrain (toppanel), midbrain (middle panel), and hindbrain/cerebellum (bottom panel)are shown.

FIG. 13. Pharmacokinetics and brain uptake of fusion protein in theadult Rhesus monkey. (A) The serum concentration, expressed as a percentof injected dose (ID)/mL, of the [¹²⁵I]-HIR Ab-IDUA fusion protein isplotted vs. time after a single intravenous injection of the protein inthe anesthetized adult Rhesus monkey; the serum concentration isexpressed as either ¹²⁵I radioactivity (closed symbol) or IDUA enzymeactivity (open symbol). (B) The volume of distribution (VD) at 120 minafter injection of the [¹²⁵I]-HIR Ab-IDUA fusion protein is shown forthe total brain homogenate and the post-vascular supernatant. Theequivalence of the VD in both compartments is evidence for transport ofthe fusion protein through the BBB in vivo (Methods). The data for the[³H]-mouse IgG2a is from Pardridge et al (1995).

FIG. 14. Genetic engineering of tandem vector (TV-HIRMAb-IDUA) encoding4 separate and tandem expression cassettes encoding the heavy chain (HC)fusion gene, the light chain (LC) gene, the DHFR gene, and the neo gene.

FIG. 15. The 3-column purification of CHO derived HIRMAb-IDUA fusionprotein uses protein A affinity chromatography (A), SP Sepharose cationexchange (CATEX) chromatography (B), and Q Sepharose anion exchange(ANEX) chromatography (C). The peak of fusion protein elution for eachcolumn is bracketed in the figure.

FIG. 16. The HIRMAb-IDUA fusion protein, derived from CHO cells, ispurified to homogeneity on reducing SDS-PAGE, as shown in lane 3. Lane 2is the chimeric HIRMAb without the fused IDUA. The MW of the HC of theHIRMAb-IDUA fusion protein is about 85 kDa larger than the HC of theHIRMAb, owing to the fusion of the IDUA enzyme. Lanes 1 and 4 are MWstandards.

FIG. 17. Western blot of the HIRMAb-IDUA fusion protein, derived fromCHO cells, using primary antibodies to either the human IgG heavy chain(lane 1) or to human IDUA (lane 2). Both antibodies react equally to the130 kDa HIRMAb-IDUA fusion protein heavy chain.

FIG. 18. Binding of either the chimeric HIRMAb or the CHO cell derivedHIRMAb-IDUA fusion protein to the HIR extracellular domain (ECD) issaturable. The ED₅₀ of HIRMAb-IDUA binding to the HIR ECD is comparableto the ED₅₀ of the binding of the chimeric HIRMAb, which indicates theaffinity for the HIR is not impaired by fusion of the IDUA to the HIRMAbheavy chain.

FIG. 19. The IDUA enzyme activity of the CHO derived HIRMAb-IDUA fusionprotein is 291±9 units/μg protein, where 1 unit=1 nmol/hr, based on afluorometric enzymatic assay that uses 4-methylumbelliferylL-α-iduronide (MUBI) as a substrate, and 4-methylumbelliferone (4-MU) asan assay standard The IDUA enzyme activity is linear with respect totime and mass of HIRMAb-IDUA fusion protein. The IDUA enzyme specificactivity of the HIRMAb-IDUA fusion protein is comparable to recombinantIDUA.

FIG. 20. Size exclusion chromatography (SEC) HPLC using 2 TosoHaasG3000SWXL columns in series. The CHO derived HIRMAb-IDUA fusion proteinelutes as a single species without aggregates.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The blood brain barrier is a severe impediment to the delivery ofsystemically administered IDUA (e.g., recombinant IDUA) to the centralnervous system. The methods and compositions described herein addressthree factors that are important in delivering a therapeuticallysignificant level of IDUA activity across the BBB to the CNS: 1)Modification of an IDUA to allow it to cross the BBB; 2) the amount andrate of uptake of systemically administered modified IDUA into the CNS,and 3) Retention of IDUA activity once across the BBB. Various aspectsof the methods and compositions described herein address these factors,by (1) providing human insulin receptor (HIR) antibody (Ab)-IDUA fusionantibodies comprising an IDUA (i.e., a protein having IDUA activity)fused, with or without intervening sequence, to an immunoglobulin (heavychain or light chain) directed against the extracellular domain of ahuman insulin receptor; and (2) establishing therapeutically effectivesystemic doses of the fusion antibodies based on a characterization oftheir uptake in the CNS and their specific activity.

Accordingly, the invention provides compositions and methods fortreating a α-L-iduronidase deficiency in the central nervous system bysystemically administering to a subject in need thereof atherapeutically effective dose of a bifunctional HIR Ab-IDUA fusionantibody having α-L-iduronidase activity and selectively binding to theextracellular domain of a human insulin receptor.

Some Definitions

“Treatment” or “treating” as used herein includes achieving atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying disorderor condition being treated. For example, in an individual with Hurler'ssyndrome, therapeutic benefit includes partial or complete halting ofthe progression of the disorder, or partial or complete reversal of thedisorder. Also, a therapeutic benefit is achieved with the eradicationor amelioration of one or more of the physiological or psychologicalsymptoms associated with the underlying condition such that animprovement is observed in the patient, notwithstanding the fact thatthe patient may still be affected by the condition. A prophylacticbenefit of treatment includes prevention of a condition, retarding theprogress of a condition (e.g., slowing the progression of a lysosomalstorage disorder), or decreasing the likelihood of occurrence of acondition. As used herein, “treating” or “treatment” includesprophylaxis.

As used herein, the term “effective amount” can be an amount, which whenadministered systemically, is sufficient to effect beneficial or desiredresults in the CNS, such as beneficial or desired clinical results, orenhanced cognition, memory, mood, or other desired CNS results. Aneffective amount is also an amount that produces a prophylactic effect,e.g., an amount that delays, reduces, or eliminates the appearance of apathological or undesired condition. Such conditions include, but arenot limited to, mental retardation, hearing loss, and neurodegeneration.An effective amount can be administered in one or more administrations.In terms of treatment, an “effective amount” of a composition of theinvention is an amount that is sufficient to palliate, ameliorate,stabilize, reverse or slow the progression of a disorder, e.g., aneurological disorder. An “effective amount” may be of any of thecompositions of the invention used alone or in conjunction with one ormore agents used to treat a disease or disorder. An “effective amount”of a therapeutic agent within the meaning of the present invention willbe determined by a patient's attending physician or veterinarian. Suchamounts are readily ascertained by one of ordinary skill in the art andwill a therapeutic effect when administered in accordance with thepresent invention. Factors which influence what a therapeuticallyeffective amount will be include, the IDUA specific activity of the HIRAb-IDUA fusion antibody administered, its absorption profile (e.g., itsrate of uptake into the brain), time elapsed since the initiation of thedisorder, and the age, physical condition, existence of other diseasestates, and nutritional status of the individual being treated.Additionally, other medication the patient may be receiving will affectthe determination of the therapeutically effective amount of thetherapeutic agent to administer.

A “subject” or an “individual,” as used herein, is an animal, forexample, a mammal. In some embodiments a “subject” or an “individual” isa human In some embodiments, the subject suffers fromMucopolysaccharidosis Type I H (“Hurler's Syndrome”),Mucopolysaccharidosis Type I S (“Scheie Syndrome”), orMucopolysaccharidosis Type I H-S (“Hurler-Scheie Syndrome).

In some embodiments, a pharmacological composition comprising anHIR-IDUA fusion antibody is “administered peripherally” or “peripherallyadministered.” As used herein, these terms refer to any form ofadministration of an agent, e.g., a therapeutic agent, to an individualthat is not direct administration to the CNS, i.e., that brings theagent in contact with the non-brain side of the blood-brain barrier.“Peripheral administration,” as used herein, includes intravenous,intra-arterial, subcutaneous, intramuscular, intraperitoneal,transdermal, by inhalation, transbuccal, intranasal, rectal, oral,parenteral, sublingual, or trans-nasal.

A “pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” herein refers to any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Such carriers are well known to those of ordinary skill inthe art. A thorough discussion of pharmaceutically acceptablecarriers/excipients can be found in Remington's Pharmaceutical Sciences,Gennaro, A R, ed., 20th edition, 2000: Williams and Wilkins Pa., USA.Exemplary pharmaceutically acceptable carriers can include salts, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. Forexample, compositions of the invention may be provided in liquid form,and formulated in saline based aqueous solution of varying pH (5-8),with or without detergents such polysothate-80 at 0.01-1%, orcarbohydrate additives, such mannitol, sorbitol, or trehalose. Commonlyused buffers include histidine, acetate, phosphate, or citrate.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally occurring amino acid, e.g., an amino acid analog. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al. J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The terms “isolated” and “purified” refer to a material that issubstantially or essentially removed from or concentrated in its naturalenvironment. For example, an isolated nucleic acid may be one that isseparated from the nucleic acids that normally flank it or other nucleicacids or components (proteins, lipids, etc. . . . ) in a sample. Inanother example, a polypeptide is purified if it is substantiallyremoved from or concentrated in its natural environment. Methods forpurification and isolation of nucleic acids and proteins are well knownin the art.

The Blood Brain Barrier

In one aspect, the invention provides compositions and methods thatutilize an IDUA fused to an HIR Ab capable of crossing the blood brainbarrier (BBB). The compositions and methods are useful in transportingIDUA from the peripheral blood and across the blood brain barrier intothe CNS. As used herein, the “blood-brain barrier” refers to the barrierbetween the peripheral circulation and the brain and spinal cord whichis formed by tight junctions within the brain capillary endothelialplasma membranes, creates an extremely tight barrier that restricts thetransport of molecules into the brain, even molecules as small as urea,molecular weight of 60 Da. The blood-brain barrier within the brain, theblood-spinal cord barrier within the spinal cord, and the blood-retinalbarrier within the retina, are contiguous capillary barriers within thecentral nervous system (CNS), and are collectively referred to as theblood-brain barrier or BBB.

The BBB limits the development of new neurotherapeutics, diagnostics,and research tools for the brain and CNS. Essentially 100% of largemolecule therapeutics such as recombinant proteins, antisense drugs,gene medicines, purified antibodies, or RNA interference (RNAi)-baseddrugs, do not cross the BBB in pharmacologically significant amounts.While it is generally assumed that small molecule drugs can cross theBBB, in fact, <2% of all small molecule drugs are active in the brainowing to the lack transport across the BBB. A molecule must be lipidsoluble and have a molecular weight less than 400 Daltons (Da) in orderto cross the BBB in pharmacologically significant amounts, and the vastmajority of small molecules do not have these dual molecularcharacteristics. Therefore, most potentially therapeutic, diagnostic, orresearch molecules do not cross the BBB in pharmacologically activeamounts. So as to bypass the BBB, invasive transcranial drug deliverystrategies are used, such as intracerebro-ventricular (ICV) infusion,intracerebral (IC) administration, and convection enhanced diffusion(CED). Transcranial drug delivery to the brain is expensive, invasive,and largely ineffective. The ICV route delivers IDUA only to theependymal surface of the brain, not into brain parenchyma, which istypical for drugs given by the ICV route. The IC administration of anenzyme such as IDUA, only provides local delivery, owing to the very lowefficiency of protein diffusion within the brain. The CED results inpreferential fluid flow through the white matter tracts of brain, whichcauses demyelination, and astrogliosis.

The methods described herein offer an alternative to these highlyinvasive and generally unsatisfactory methods for bypassing the BBB,allowing a functional IDUA to cross the BBB from the peripheral bloodinto the CNS following systemic administration of an HIR-IDUA fusionantibody composition described herein. The methods described hereinexploit the expression of insulin receptors (e g, human insulinreceptors) on the BBB to shuttle desired a bifunctional HIR-IDUA fusionantibody from peripheral blood into the CNS.

Insulin Receptors

The BBB has been shown to have specific receptors, including insulinreceptors, that allow the transport from the blood to the brain ofseveral macromolecules. In particular, insulin receptors are suitable astransporters for the HIR Ab-IDUA fusion antibodies described herein. TheHIR-IDUA fusion antibodies described herein bind to the extracellulardomain (ECD) of the human insulin receptor.

Insulin receptors and their extracellular, insulin binding domain (ECD)have been extensively characterized in the art both structurally andfunctionally. See, e.g., Yip et al (2003), “J Biol. Chem,278(30):27329-27332; and Whittaker et al. (2005), J Biol Chem,280(22):20932-20936. The amino acid and nucleotide sequences of thehuman insulin receptor can be found under GenBank accession No.NM_000208.

Antibodies that Bind to an Insulin Receptor Mediated Transport Systemand Other Receptor Mediated Transport Systems of the BBB

One noninvasive approach for the delivery of IDUA to the CNS is to fusethe IDUA to an antibody that selectively binds to the ECD of the insulinreceptor. Insulin receptors expressed on the BBB can thereby serve as avector for transport of the IDUA across the BBB. Certain ECD-specificantibodies may mimic the endogenous ligand and thereby traverse a plasmamembrane barrier via transport on the specific receptor system. Suchinsulin receptor antibodies act as molecular “Trojan horses,” asdepicted schematically in FIG. 7. Thus, despite the fact that antibodiesand other macromolecules are normally excluded from the brain, they canbe an effective vehicle for the delivery of molecules into the brainparenchyma if they have specificity for the extracellular domain of areceptor expressed on the BBB, e.g., the insulin receptor. In certainembodiments, an HIR Ab-IDUA fusion antibody binds an exofacial epitopeon the human BBB HIR and this binding enables the fusion antibody totraverse the BBB via a transport reaction that is mediated by the humanBBB insulin receptor. In addition, other known BBB targeting agents(also referred to herein as “Trojan horses”), such as endogenouspeptides or modified proteins, including endogenous peptides, such astransferrin, insulin, leptin, insulin-like growth factors (IGFs), orcationic peptides, or peptidomimetic monoclonal antibodies to the BBBtransferrin receptor, insulin receptor, IGF receptor, or leptin receptormay be used to deliver enzymes of the type and size-range mentionedabove across the BBB.

The term “antibody” describes an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anypolypeptide or protein having a binding domain which is, or ishomologous to, an antigen-binding domain CDR grafted antibodies are alsocontemplated by this term.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains Each lightchain is typically linked to a heavy chain by one covalent disulfidebond, while the number of disulfide linkages varies among the heavychains of different immunoglobulin isotypes. Each heavy and light chainalso has regularly spaced intrachain disulfide bridges. Each heavy chainhas at one end a variable domain (“VH”) followed by a number of constantdomains (“CH”). Each light chain has a variable domain at one end (“VL”)and a constant domain (“CL”) at its other end; the constant domain ofthe light chain is aligned with the first constant domain of the heavychain, and the light-chain variable domain is aligned with the variabledomain of the heavy chain Particular amino acid residues are believed toform an interface between the light- and heavy-chain variable domains.

The term “variable domain” refers to protein domains that differextensively in sequence among family members (i.e. among differentisoforms, or in different species). With respect to antibodies, the term“variable domain” refers to the variable domains of antibodies that areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the “framework region” or “FR”. The variabledomains of unmodified heavy and light chains each comprise four FRs(FR1, FR2, FR3 and FR4, respectively), largely adopting a γ-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991), pages 647-669). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from three“complementarity determining regions” or “CDRs”, which directly bind, ina complementary manner, to an antigen and are known as CDR1, CDR2, andCDR3 respectively.

In the light chain variable domain, the CDRs typically correspond toapproximately residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3),and in the heavy chain variable domain the CDRs typically correspond toapproximately residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3);Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901 917 (1987)).

As used herein, “variable framework region” or “VFR” refers to frameworkresidues that form a part of the antigen binding pocket or groove and/orthat may contact antigen. In some embodiments, the framework residuesform a loop that is a part of the antigen binding pocket or groove. Theamino acids residues in the loop may or may not contact the antigen. Inan embodiment, the loop amino acids of a VFR are determined byinspection of the three-dimensional structure of an antibody, antibodyheavy chain, or antibody light chain. The three-dimensional structurecan be analyzed for solvent accessible amino acid positions as suchpositions are likely to form a loop and/or provide antigen contact in anantibody variable domain Some of the solvent accessible positions cantolerate amino acid sequence diversity and others (e.g. structuralpositions) can be less diversified. The three dimensional structure ofthe antibody variable domain can be derived from a crystal structure orprotein modeling. In some embodiments, the VFR comprises, consistessentially of, or consists of amino acid positions corresponding toamino acid positions 71 to 78 of the heavy chain variable domain, thepositions defined according to Kabat et al., 1991. In some embodiments,VFR forms a portion of Framework Region 3 located between CDRH2 andCDRH3. The VFR can form a loop that is well positioned to make contactwith a target antigen or form a part of the antigen binding pocket.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains (Fc) that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa or (“κ”) and lambda or (“λ”), based on the amino acid sequences oftheir constant domains.

In referring to an antibody or fusion antibody described herein, theterms “selectively bind,” “selectively binding,” “specifically binds,”or “specifically binding” refer to binding to the antibody or fusionantibody to its target antigen for which the dissociation constant (Kd)is about 10⁻⁶ M or lower, i.e., 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or10⁻¹²M.

The term antibody as used herein will also be understood to mean one ormore fragments of an antibody that retain the ability to specificallybind to an antigen, (see generally, Holliger et al., Nature Biotech. 23(9) 1126-1129 (2005)). Non-limiting examples of such antibodies include(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region;(iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544 546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423 426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879 5883; and Osbourn et al. (1998) Nat.Biotechnol. 16:778). Such single chain antibodies are also intended tobe encompassed within the term antibody. Any VH and VL sequences ofspecific scFv can be linked to human immunoglobulin constant region cDNAor genomic sequences, in order to generate expression vectors encodingcomplete IgG molecules or other isotypes. VH and VL can also be used inthe generation of Fab, Fv or other fragments of immunoglobulins usingeither protein chemistry or recombinant DNA technology. Other forms ofsingle chain antibodies, such as diabodies are also encompassed.

“F(ab′)2” and “Fab′” moieties can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andincludes an antibody fragment generated by digesting immunoglobulin nearthe disulfide bonds existing between the hinge regions in each of thetwo H chains. For example, papain cleaves IgG upstream of the disulfidebonds existing between the hinge regions in each of the two H chains togenerate two homologous antibody fragments in which an L chain composedof VL (L chain variable region) and CL (L chain constant region), and anH chain fragment composed of VH (H chain variable region) and CHyl (ylregion in the constant region of H chain) are connected at their Cterminal regions through a disulfide bond. Each of these two homologousantibody fragments is called Fab′. Pepsin also cleaves IgG downstream ofthe disulfide bonds existing between the hinge regions in each of thetwo H chains to generate an antibody fragment slightly larger than thefragment in which the two above-mentioned Fab′ are connected at thehinge region. This antibody fragment is called F(ab′)2.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

“Single-chain Fv” or “sFv” antibody fragments comprise a VH, a VL, orboth a VH and VL domain of an antibody, wherein both domains are presentin a single polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the VH and VL domainswhich enables the sFv to form the desired structure for antigen binding.For a review of sFv see, e.g., Pluckthun in The Pharmacology ofMonoclonal Antibodies, Vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269 315 (1994).

A “chimeric” antibody includes an antibody derived from a combination ofdifferent mammals The mammal may be, for example, a rabbit, a mouse, arat, a goat, or a human. The combination of different mammals includescombinations of fragments from human and mouse sources.

In some embodiments, an antibody of the present invention is amonoclonal antibody (MAb), typically a chimeric human-mouse antibodyderived by humanization of a mouse monoclonal antibody. Such antibodiesare obtained from, e.g., transgenic mice that have been “engineered” toproduce specific human antibodies in response to antigenic challenge. Inthis technique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesis human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas.

For use in humans, a chimeric HIR Ab is preferred that contains enoughhuman sequence that it is not significantly immunogenic whenadministered to humans, e.g., about 80% human and about 20% mouse, orabout 85% human and about 15% mouse, or about 90% human and about 10%mouse, or about 95% human and 5% mouse, or greater than about 95% humanand less than about 5% mouse. Chimeric antibodies to the human BBBinsulin receptor with sufficient human sequences for use in theinvention are described in, e.g., Boado et al. (2007), BiotechnolBioeng, 96(2):381-391. A more highly humanized form of the HIR MAb canalso be engineered, and the humanized HIR Ab has activity comparable tothe murine HIR Ab and can be used in embodiments of the invention. See,e.g., U.S. Patent Application Publication Nos. 20040101904, filed Nov.27, 2002 and 20050142141, filed Feb. 17, 2005.

In exemplary embodiments, the HIR antibodies or HIR-IDUA fusionantibodies derived therefrom contain an immunoglobulin heavy chaincomprising CDRs corresponding to the sequence of at least one of the HCCDRs listed in FIG. 3 (SEQ ID NOs 1-3) or a variant thereof. Forexample, a HC CDR1 corresponding to the amino acid sequence of SEQ IDNO:1 with up to 1, 2, 3, 4, 5, or 6 single amino acid mutations, a HCCDR2 corresponding to the amino acid sequence of SEQ ID NO:2 with up to1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single amino acid mutations, or a HCCDR3 corresponding to the amino acid sequence of SEQ ID NO:3 with up to1, or 2 single amino acid mutations, where the single amino acidmutations are substitutions, deletions, or insertions.

In other embodiments, the HIR Abs or HIR Ab-IDUA fusion Abs contain animmunoglobulin HC the amino acid sequence of which is at least 50%identical (i.e., at least, 55, 60, 65, 70, 75, 80, 85, 90, 95, or anyother percent up to 100% identical) to SEQ ID NO:7 (shown in FIG. 1).

In some embodiments, the HIR Abs or HIR AB-IDUA fusion Abs include animmunoglobulin light chain comprising CDRs corresponding to the sequenceof at least one of the LC CDRs listed in FIG. 3 (SEQ ID NOs: 4-6) or avariant thereof. For example, a LC CDR1 corresponding to the amino acidsequence of SEQ ID NO:4 with up to 1, 2, 3, 4, or 5 single amino acidmutations, a LC CDR2 corresponding to the amino acid sequence of SEQ IDNO:5 with up to 1, 2, 3, or 4 single amino acid mutations, or a LC CDR3corresponding to the amino acid sequence of SEQ ID NO:6 with up to 1, 2,3, 4, or 5 single amino acid mutations.

In other embodiments, the HIR Abs or HIR AB-IDUA fusion Abs contain animmunoglobulin LC the amino acid sequence of which is at least 50%identical (i.e., at least, 55, 60, 65, 70, 75, 80, 85, 90, 95, or anyother percent up to 100% identical) to SEQ ID NO:8 (shown in FIG. 2).

In yet other embodiments, the HIR Abs or HIR Ab-IDUA fusion Abs containboth a heavy chain and a light chain corresponding to any of theabove-mentioned HIR heavy chains and HIR light chains.

HIR antibodies used in the invention may be glycosylated ornon-glycosylated. If the antibody is glycosylated, any pattern ofglycosylation that does not significantly affect the function of theantibody may be used. Glycosylation can occur in the pattern typical ofthe cell in which the antibody is made, and may vary from cell type tocell type. For example, the glycosylation pattern of a monoclonalantibody produced by a mouse myeloma cell can be different than theglycosylation pattern of a monoclonal antibody produced by a transfectedChinese hamster ovary (CHO) cell. In some embodiments, the antibody isglycosylated in the pattern produced by a transfected Chinese hamsterovary (CHO) cell.

One of ordinary skill in the art will appreciate that currenttechnologies permit a vast number of sequence variants of candidate HIRAbs or known HIR Abs to be readily generated be (e.g., in vitro) andscreened for binding to a target antigen such as the ECD of the humaninsulin receptor or an isolated epitope thereof. See, e.g., Fukuda etal. (2006) “In vitro evolution of single-chain antibodies using mRNAdisplay,” Nuc. Acid Res., 34(19) (published online) for an example ofultra high throughput screening of antibody sequence variants. See also,Chen et al. (1999), “In vitro scanning saturation mutagenesis of all thespecificity determining residues in an antibody binding site,” Prot Eng,12(4): 349-356. An insulin receptor ECD can be purified as described in,e.g., Coloma et al. (2000) Pharm Res, 17:266-274, and used to screen forHIR Abs and HIR Ab sequence variants of known HIR Abs.

Accordingly, in some embodiments, a genetically engineered HIR Ab, withthe desired level of human sequences, is fused to an IDUA, to produce arecombinant fusion antibody that is a bi-functional molecule. The HIRAb-IDUA fusion antibody: (i) binds to an extracellular domain of thehuman insulin receptor; (ii) catalyzes hydrolysis of unsulfatedalpha-L-iduronosidic linkages in dermatan sulfate; and (iii) is able tocross the BBB, via transport on the BBB HIR, and retain IDUA activityonce inside the brain, following peripheral administration.

α-L-Iduronidase (IDUA)

Systemic administration (e.g., by intravenous injection) of recombinantIDUA (e.g., Aldurazyme®) fails to rescue a deficiency of IDUA in the CNSof patients suffering from Hurler's syndrome. IDUA does not cross theBBB, and the lack of transport of the enzyme across the BBB prevents itfrom having a significant therapeutic effect in the CNS followingperipheral administration. However, when the IDUA is fused to an HIR Ab(e.g., by a linker), this enzyme is now able to enter the CNS from bloodfollowing a non-invasive peripheral route of administration such asintravenous, intra-arterial, intramuscular, subcutaneous,intraperitoneal, or even oral administration Administration of a HIRAb-IDUA fusion antibody enables delivery of IDUA activity into the brainfrom peripheral blood. Described herein is the determination of asystemic dose of the HIR Ab-IDUA fusion antibody that is therapeuticallyeffective for treating an IDUA deficiency in the CNS. As describedherein, appropriate systemic doses of an HIR Ab-IDUA fusion antibody areestablished based on a quantitative determination of CNS uptakecharacteristics and enzymatic activity of an HIR Ab-IDUA fusionantibody.

As used herein, IDUA refers to any naturally occurring or artificialenzyme that can catalyze the hydrolysis of unsulfatedalpha-L-iduronosidic linkages in dermatan sulfate, e.g., the human IDUAsequence listed under GenBank Accession No. NP 000194.

In some embodiments, IDUA has an amino acid sequence that is a at least50% identical (i.e., at least, 55, 60, 65, 70, 75, 80, 85, 90, 95, orany other percent up to 100% identical) to the amino acid sequence ofhuman IDUA, a 653 amino acid protein listed under GenBank Accession No.NP_000194, or a 627 amino acid subsequence thereof, which lacks a 26amino acid signal peptide, and corresponds to SEQ ID NO:9 (FIG. 4). Thestructure-function relationship of human IDUA is well established, asdescribed in, e.g., Rempel et al. (2005), “A homology model for humanα-L-Iduronidase: Insights into human disease,” Mol. Genetics and Met.,85:28-37. In particular, residues that are critical to the function ofIDUA include, e.g., Gly 51, Ala 75, Ala 160, Glu 182, Gly 208, Leu 218,Asp 315, Ala 327, Asp 349, Thr 366, Thr 388, Arg 489, Arg 628, Ala 79,His 82, Glu 178, Ser 260, Leu 346, Asn 350, Thr 364, Leu 490, Pro 496,Pro 533, Arg 619, Arg 89, Cys 205, His 240, Ala 319, Gln 380, Arg 383,and Arg 492.

In some embodiments, IDUA has an amino acid sequence at least 50%identical (i.e., at least, 55, 60, 65, 70, 75, 80, 85, 90, 95, or anyother percent up to 100% identical) to SEQ ID NO:9 (shown in FIG. 4).Sequence variants of a canonical IDUA sequence such as SEQ ID NO:9 canbe generated, e.g., by random mutagenesis of the entire sequence orspecific subsequences corresponding to particular domains.Alternatively, site directed mutagenesis can be performed reiterativelywhile avoiding mutations to residues known to be critical to IDUAfunction such as those given above. Further, in generating multiplevariants of an IDUA sequence, mutation tolerance prediction programs canbe used to greatly reduce the number of non-functional sequence variantsthat would be generated by strictly random mutagenesis. Variousprograms) for predicting the effects of amino acid substitutions in aprotein sequence on protein function (e.g., SIFT, PolyPhen, PANTHERPSEC, PMUT, and TopoSNP) are described in, e.g., Henikoff et al. (2006),“Predicting the Effects of Amino Acid Substitutions on ProteinFunction,” Annu. Rev. Genomics Hum. Genet., 7:61-80. IDUA sequencevariants can be screened for of IDUA activity/retention of IDUA activityby, e.g., 4-methylumbelliferyl α-L-iduronide (MUBI) flurometric IDUAassays known in the art. See, e.g., Kakkis et al. (1994), Prot ExprPurif 5:225-232. One unit of IDUA activity is defined as the hydrolysisof 1 nmole substrate/hour. Accordingly, one of ordinary skill in the artwill appreciate that a very large number of operable IDUA sequencevariants can be obtained by generating and screening extremely diverse“libraries” of IDUA sequence variants by methods that are routine in theart, as described above.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.). Thepercent identity is then calculated as: ([Total number of identicalmatches]/[length of the longer sequence plus the number of gapsintroduced into the longer sequence in order to align the twosequences])(100).

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of anotherpeptide. The FASTA algorithm is described by Pearson and Lipman, Proc.Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol.183:63 (1990). Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:24 orSEQ ID NO: 39) and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

The present invention also includes proteins having a conservative aminoacid change, compared with an amino acid sequence disclosed herein.Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. The BLOSUM62 table is an aminoacid substitution matrix derived from about 2,000 local multiplealignments of protein sequence segments, representing highly conservedregions of more than 500 groups of related proteins (Henikoff andHenikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, theBLOSUM62 substitution frequencies can be used to define conservativeamino acid substitutions that may be introduced into the amino acidsequences of the present invention. Although it is possible to designamino acid substitutions based solely upon chemical properties (asdiscussed above), the language “conservative amino acid substitution”preferably refers to a substitution represented by a BLOSUM62 value ofgreater than −1. For example, an amino acid substitution is conservativeif the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or3. According to this system, preferred conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 1 (e.g.,1, 2 or 3), while more preferred conservative amino acid substitutionsare characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

It also will be understood that amino acid sequences may includeadditional residues, such as additional N- or C-terminal amino acids,and yet still be essentially as set forth in one of the sequencesdisclosed herein, so long as the sequence retains sufficient biologicalprotein activity to be functional in the compositions and methods of theinvention.

Compositions

Strikingly, it has been found that the bifunctional HIR Ab-IDUA fusionantibodies described herein, retain a high proportion of the activity oftheir separate constituent proteins, i.e., binding of the HIR Ab to theIR ECD and transport across the BBB, and the enzymatic activity of IDUA.Construction of cDNAs and expression vectors encoding any of theproteins described herein, as well as their expression and purificationare well within those of ordinary skill in the art, and are described indetail herein in, e.g., Examples 1-3, and, in Boado et al (2007),Biotechnol Bioeng 96:381-391, U.S. patent application Ser. No.11/061,956, and U.S. patent application Ser. No. 11/245,710.

Described herein are bifunctional HIR Ab-IDUA fusion antibodiescontaining a HIR Ab, as described herein, capable of crossing the BBBfused to IDUA, where the HIR Ab is capable of crossing the blood brainbarrier and the IDUA each retain an average of at least about 10, 20,30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a HIR Ab-IDUA fusion antibody where the HIR Aband IDUA each retain an average of at least about 50% of theiractivities, compared to their activities as separate entities. In someembodiments, the invention provides a HIR Ab-IDUA fusion antibody wherethe HIR Ab and IDUA each retain an average of at least about 60% oftheir activities, compared to their activities as separate entities. Insome embodiments, the invention provides a HIR Ab-IDUA fusion antibodywhere the HIR Ab and IDUA each retain an average of at least about 70%of their activities, compared to their activities as separate entities.In some embodiments, the invention provides a HIR Ab-IDUA fusionantibody where the HIR Ab and IDUA each retain an average of at leastabout 80% of their activities, compared to their activities as separateentities. In some embodiments, the invention provides a fusion HIRAb-IDUA fusion antibody where the HIR Ab and IDUA each retain an averageof at least about 90% of their activities, compared to their activitiesas separate entities. In some embodiments, the HIR Ab retains at leastabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of itsactivity, compared to its activity as a separate entity, and the IDUAretains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or100% of its activity, compared to its activity as a separate entity.Accordingly, described herein are compositions containing a bifunctionalHIR Ab-IDUA fusion antibody capable of crossing the BBB, where theconstituent HIR Ab and IDUA each retain, as part of the fusion antibody,an average of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99,or 100% of their activities, i.e., HIR binding and IDUA activity,respectively, compared to their activities as separate proteins. An HIRAb IDUA fusion antibody refers to a fusion protein comprising any of theHIR antibodies and IDUAs described herein.

In the HIR Ab-IDUA fusion antibodies described herein, the covalentlinkage between the antibody and the IDUA may be to the carboxy or aminoterminal of the HIR antibody and the amino or carboxy terminal of theIDUA as long as the linkage allows the HIR Ab-IDUA fusion antibody tobind to the ECD of the IR and cross the blood brain barrier, and allowsthe IDUA to retain a therapeutically useful portion of its activity. Incertain embodiments, the covalent link is between an HC of the antibodyand the IDUA or a LC of the antibody and the IDUA. Any suitable linkagemay be used, e.g., carboxy terminus of light chain to amino terminus ofIDUA, carboxy terminus of heavy chain to amino terminus of IDUA, aminoterminus of light chain to amino terminus of IDUA, amino terminus ofheavy chain to amino terminus of IDUA, carboxy terminus of light chainto carboxy terminus of IDUA, carboxy terminus of heavy chain to carboxyterminus of IDUA, amino terminus of light chain to carboxy terminus ofIDUA, or amino terminus of heavy chain to carboxy terminus of IDUA. Insome embodiments, the linkage is from the carboxy terminus of the HC tothe amino terminus of the IDUA.

It will be appreciated that a linkage between terminal amino acids canbe accomplished by an intervening peptide linker sequence that formspart of the fused amino acid sequence. The peptide sequence linker maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acids in length.In some embodiments, a two amino acid linker is used. In someembodiments, the linker has the sequence ser-ser. The peptide linkersequence may include a protease cleavage site, however this is not arequirement for activity of the IDUA; indeed, an advantage of theseembodiments of the present invention is that the bifunctional HIRAb-IDUA fusion antibody, without cleavage, is partially or fully activeboth for transport and for activity once across the BBB. FIG. 5 shows anexemplary embodiment of the amino acid sequence of a HIR Ab-IDUA fusionantibody (SEQ ID NO:10) in which the HC is fused through its carboxyterminus via a two amino acid “ser-ser” linker to the amino terminus ofthe IDUA. In some embodiments, the fused IDUA sequence is devoid of its26 amino acid signal peptide, as shown in FIG. 4.

In some embodiments, a HIR Ab-IDUA fusion antibody comprises both a HCand a LC. In some embodiments, the HIR Ab-IDUA fusion antibody is amonovalent antibody. In other embodiments, the HIR Ab-IDUA fusionantibody is a divalent antibody, as described herein in the Examplesection.

The HIR Ab used as part of the HIR Ab-IDUA fusion antibody can beglycosylated or nonglycosylated; in some embodiments, the antibody isglycosylated, e.g., in a glycosylation pattern produced by its synthesisin a CHO cell.

As used herein, “activity” includes physiological activity (e.g.,ability to cross the BBB and/or therapeutic activity), binding affinityof the HIR Ab for the IR ECD, or the enzymatic activity of IDUA.

Transport of a HIR Ab-IDUA fusion antibody across the BBB may becompared to transport across the BBB of the HIR Ab alone by standardmethods. For example, pharmacokinetics and brain uptake of the HIRAb-IDUA fusion antibody by a model animal, e.g., a mammal such as aprimate, may be used. Such techniques are illustrated in Example 5,which demonstrates pharmacokinetics and brain uptake of a fusion proteinof the invention by the adult Rhesus monkey. Similarly, standard modelsfor determining IDUA activity may also be used to compare the functionof the IDUA alone and as part of a HIR Ab-IDUA fusion antibody. See,e.g., Example 3, which demonstrates the enzymatic activity of IDUAversus HIR Ab-IDUA fusion antibody. Binding affinity for the IR ECD canbe compared for the HIR Ab-IDUA fusion antibody versus the HIR Ab alone.See, e.g., Example 4 herein.

Also included herein are pharmaceutical compositions that contain one ormore HIR Ab-IDUA fusion antibodies described herein and apharmaceutically acceptable excipient. A thorough discussion ofpharmaceutically acceptable carriers/excipients can be found inRemington's Pharmaceutical Sciences, Gennaro, A R, ed., 20th edition,2000: Williams and Wilkins Pa., USA. Pharmaceutical compositions of theinvention include compositions suitable for administration via anyperipheral route, including intravenous, subcutaneous, intramuscular,intraperitoneal injection; oral, rectal, transbuccal, pulmonary,transdermal, intranasal, or any other suitable route of peripheraladministration.

The compositions of the invention are particular suited for injection,e.g., as a pharmaceutical composition for intravenous, subcutaneous,intramuscular, or intraperitoneal administration. Aqueous compositionsof the present invention comprise an effective amount of a compositionof the present invention, which may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, e g, a human, asappropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Exemplary pharmaceutically acceptable carriers for injectablecompositions can include salts, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. For example, compositions of the invention maybe provided in liquid form, and formulated in saline based aqueoussolution of varying pH (5-8), with or without detergents suchpolysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol,sorbitol, or trehalose. Commonly used buffers include histidine,acetate, phosphate, or citrate. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol; phenol, sothic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate, and gelatin.

For human administration, preparations meet sterility, pyrogenicity,general safety, and purity standards as required by FDA and otherregulatory agency standards. The active compounds will generally beformulated for parenteral administration, e.g., formulated for injectionvia the intravenous, intramuscular, subcutaneous, intralesional, orintraperitoneal routes. The preparation of an aqueous composition thatcontains an active component or ingredient will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use in preparing solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation include vacuum-drying and freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be systemically administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective based on the criteria described herein. Theformulations are easily administered in a variety of dosage forms, suchas the type of injectable solutions described above, but drug releasecapsules and the like can also be employed

The appropriate quantity of a pharmaceutical composition to beadministered, the number of treatments, and unit dose will varyaccording to the CNS uptake characteristics of a HIR Ab-IDUA fusionantibody as described herein, and according to the subject to betreated, the state of the subject and the effect desired. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other alternativemethods of administration of the present invention may also be used,including but not limited to intradermal administration (See U.S. Pat.Nos. 5,997,501; 5,848,991; and 5,527,288), pulmonary administration (SeeU.S. Pat. Nos. 6,361,760; 6,060,069; and 6,041,775), buccaladministration (See U.S. Pat. Nos. 6,375,975; and 6,284,262),transdermal administration (See U.S. Pat. Nos. 6,348,210; and 6,322,808)and transmucosal administration (See U.S. Pat. No. 5,656,284). Suchmethods of administration are well known in the art. One may also useintranasal administration of the present invention, such as with nasalsolutions or sprays, aerosols or inhalants Nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions. Thus, the aqueous nasal solutionsusually are isotonic and slightly buffered to maintain a pH of 5.5 to6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations and appropriate drug stabilizers, if required,may be included in the formulation. Various commercial nasalpreparations are known and include, for example, antibiotics andantihistamines and are used for asthma prophylaxis.

Additional formulations, which are suitable for other modes ofadministration, include suppositories and pessaries. A rectal pessary orsuppository may also be used. Suppositories are solid dosage forms ofvarious weights and shapes, usually medicated, for insertion into therectum or the urethra. After insertion, suppositories soften, melt ordissolve in the cavity fluids. For suppositories, traditional bindersand carriers generally include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in any suitable range, e.g., in the range of 0.5%to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders. Incertain defined embodiments, oral pharmaceutical compositions willcomprise an inert diluent or assimilable edible carrier, or they may beenclosed in a hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations can contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried, and may conveniently be between about 2 to about 75% of theweight of the unit, or between about 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, such as gum tragacanth, acacia, cornstarch, orgelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup of elixir may contain the active compoundssucrose as a sweetening agent, methylene and propyl parabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Insome embodiments, an oral pharmaceutical composition may be entericallycoated to protect the active ingredients from the environment of thestomach; enteric coating methods and formulations are well-known in theart.

Methods

Described herein are methods for delivering an effective dose of IDUA tothe CNS across the BBB by systemically administering a therapeuticallyeffective amount of a HIR Ab-IDUA fusion antibody, as described herein.Suitable systemic doses for delivery of a HIR Ab-IDUA fusion antibody isbased on its CNS uptake characteristics and IDUA specific activity asdescribed herein. Systemic administration of a HIR Ab-IDUA fusionantibody to a subject suffering from an IDUA deficiency is an effectiveapproach to the non-invasive delivery of IDUA to the CNS.

The amount of a HIR-IDUA fusion antibody that is a therapeuticallyeffective systemic dose of a HIR Ab-IDUA fusion antibody depends, inpart, on the CNS uptake characteristics of the HIR-IDUA fusion antibodyto be administered, as described herein, e.g., the percentage of thesystemically administered dose to be taken up in the CNS,

In some embodiments, 0.3% (i.e., about 0.32%, 0.4%, 0.48%, 0.6%, 0.74%,0.8%, 0.9%, 1.05, 1.1, 1.2, 1.3%, 1.5%, 2%, 2.5%, 5%, or any % fromabout 0.3% to about 12%) of the systemically administered HIR Ab-IDUAfusion antibody is delivered to the brain as a result of its uptake fromperipheral blood across the BBB. In some embodiments, at least 0.5%,(i.e., about 0.32%, 0.4%, 0.48%, 0.6%, 0.74%, 0.8%, 0.9%, 1.05, 1.1,1.2, 1.3%, 1.5%, 2%, 2.5%, 5%, or any % from about 0.3% to about 12%) ofthe systemically administered dose of the HIR Ab-IDUA fusion antibody isdelivered to the brain within two hours or less, i.e., 1.8, 1.7, 1.5,1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.6, 0.5 or any other period from about0.5 to about two hours after systemic administration.

Accordingly, in some embodiments the invention provides methods ofadministering a therapeutically effective amount of a HIR Ab-IDUA fusionantibody systemically, such that the amount of the HIR Ab-IDUA fusionantibody to cross the BBB provides at least 0.2 units of IDUAactivity/mg protein in the subject's brain, i.e., 0.21, 0.22, 0.25, 0.4,0.5, 0.6, 0.7, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 2, 2.2, 2.4,2.5, 2.7, 2.8, 3, 4, or any other value from 0.2 to 4 of units of IDUAactivity/mg protein in the subject's brain.

In some embodiments, the total number of units of IDUA activitydelivered to a subject's brain is at least, 25,000 units, i.e., at least25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000,90,000, 110,000, 120,000, 130,000, 140,000, 200,000, 210,000, 220,000,230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 300,000 or anyother total number of IDUA units from about 25,000 to 300,000 units ofIDUA activity.

In some embodiments, a therapeutically effective systemic dose comprisesat least 5×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4, 10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶,9×10⁶, 1×10⁷, 1.1×10⁷, 1.2×10⁷, 1.5×10⁷, 1.6×10⁷, 1.7×10⁷, 1.8×10⁷,1.9×10⁷, 2×10⁷, 2.1×10⁷, 3×10⁷, or any other systemic dose from about5×10⁵ to 3×10⁷ units of IDUA activity.

In other embodiments, a therapeutically effective systemic dose is atleast about 100,000 units of IDUA activity/kg body weight, i.e., atleast about 110,000, 120,000, 130,000, 140,000, 200,000, 210,000,220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 300,000or any other number of IDUA units from about 110,000 to 300,000 units ofIDUA activity/kg of body weight.

One of ordinary skill in the art will appreciate that the mass amount ofa therapeutically effective systemic dose of a HIR Ab-IDUA fusionantibody will depend, in part, on its IDUA specific activity. In someembodiments, the IDUA specific activity of a HIR Ab-IDUA fusion antibodyis at least 100,000 U/mg of protein, i.e., at least about 110,000,120,000, 130,000, 140,000, 200,000, 210,000, 220,000, 230,000, 240,000,250,000, 260,000, 270,000, 280,000, 300,000, 320,000, 340,000, 350,000,360,000, 370,000, 373,000, 400,000, 500,000, or any other specificactivity value from about 100,000 units/mg to about 500,000 units/mg.

Thus, with due consideration of the specific activity of a HIR Ab-IDUAfusion antibody and the body weight of a subject to be treated, asystemic dose of the HIR Ab-IDUA fusion antibody can be at least 2 mg,i.e., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, orany other value from about 2 mg to about 100 mg of HIR Ab-IDUA fusionantibody.

The term “systemic administration” or “peripheral administration,” asused herein, includes any method of administration that is not directadministration into the CNS, i.e., that does not involve physicalpenetration or disruption of the BBB. “Systemic administration”includes, but is not limited to, intravenous, intra-arterialintramuscular, subcutaneous, intraperitoneal, intranasal, transbuccal,transdermal, rectal, transalveolar (inhalation), or oral administration.Any suitable HIR Ab-IDUA fusion antibody, as described herein, may beused.

An IDUA deficiency as referred to herein includes, one or moreconditions known as Hurler's syndrome, Hurler's disease,mucopolysaccharidosis type I, Scheie syndrome (MPS I S), andHurler-Scheie (MPS I H-S). The IDUA deficiency is characterized by thebuildup of heparan sulfate and dermatan sulfate occurs in the body (theheart, liver, brain etc.).

The compositions of the invention, i.e., an HIR Ab-IDUA fusion antibodymay be administered as part of a combination therapy. The combinationtherapy involves the administration of a composition of the invention incombination with another therapy for treatment or relief of symptomstypically found in a patient suffering from an IDUA deficiency. If thecomposition of the invention is used in combination with another CNSdisorder method or composition, any combination of the composition ofthe invention and the additional method or composition may be used.Thus, for example, if use of a composition of the invention is incombination with another CNS disorder treatment agent, the two may beadministered simultaneously, consecutively, in overlapping durations, insimilar, the same, or different frequencies, etc. In some cases acomposition will be used that contains a composition of the invention incombination with one or more other CNS disorder treatment agents.

In some embodiments, the composition, e.g., an HIR Ab-IDUA fusionantibody is co-administered to the patient with another medication,either within the same formulation or as a separate composition. Forexample, the HIR Ab-IDUA fusion antibody could be formulated withanother fusion protein that is also designed to deliver across the humanblood-brain barrier a recombinant protein other than IDUA. Further, thefusion HIR Ab-IDUA fusion antibody may be formulated in combination withother large or small molecules.

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. All publications cited hereinare hereby incorporated by reference in their entirety. Where referenceis made to a URL or other such identifier or address, it is understoodthat such identifiers can change and particular information on theinternet can come and go, but equivalent information can be found bysearching the internet. Reference thereto evidences the availability andpublic dissemination of such information.

Example 1 Construction of Human HIR Ab Heavy Chain-IDUA Fusion ProteinExpression Vector

Human IDUA cDNA corresponding to amino acids Met1-Pro653 of the maturehuman IDUA protein, including the 26 amino acid signal peptide(NP_00194), was cloned by reverse transcription (RT) polymerase chainreaction (PCR) using the oligodeoxynucleotides (ODNs) listed in Table 1,designated ‘IDUA forward primer’ and ‘IDUA reverse primer’, and humanliver polyA+ RNA (Clontech). Human liver cDNA was prepared using theSuper Script first-strand synthesis kit (Invitrogen, San Diego, Calif.)and oligodeoxythymidine priming as per the manufacturer's instructions.The IDUAcDNA was cloned by PCR using 2 μl liver cDNA reversetranscription reaction, 0.2 μM IDUA forward and reverse ODN primers(Table 1), 0.2 mM dNTPs and 2.5 U PfuUltraDNA polymerase (Stratagene,San Diego, Calif.) in a 50 μl Pfu buffer (Stratagene). The amplificationwas performed in a Master cycler temperature cycler (Eppendorf, Hamburg,Germany) with an initial denaturing step of 95° C. for 2 min followed by30 cycles of denaturing at 95° C. for 30 sec, annealing at 55° C. for 30sec and amplification at 72° C. for 1 min. PCR products were resolved in1% agarose gel electrophoresis, and the expected major single band of−1.9 kb corresponding to the human IDUA cDNA was isolated (FIG. 8). Thecloned human IDUA was inserted into the pcDNA eukaryotic expressionplasmid at the EcoRV site, and this IDUA expression plasmid wasdesignated pCD-IDUA. The entire expression cassette of the plasmid wasconfirmed by sequencing both strands.

TABLE 1 Nucleotide Oligodeoxynucleotide primers used inthe RT-PCR cloning of human IDUA IDUA FORWARD PRIMER:5′-phosphate-GCGTGGCCATGCGTCCCCTGCGCCCCCGCGCCGCGCTGCTGGCGCTCCTG (SEQ ID NO: 11) IDUA-signal peptide FORWARD PRIMER:5′-phosphate-CAGAGGCCCCGCACCTGGTGCAGGTGGACGCGGCCCGCGCGCTGTG (SEQ ID NO: 12) IDUA REVERSE PRIMER:5′-phosphate-TCATGGATTGCCCGGGGATGGGGGCCCTCTTGGCACAGGGACC (SEQ ID NO: 13)

DNA sequencing of the expression cassette of the pCD-IDUA encompassed3,085 nucleotides (nt), including a 715 nt CMV promoter, a 1,962 nt IDUAopen reading frame, and a 401 nt BGH sequence, which predicted a 653amino acid human IDUA protein, including a 26 amino acid signal peptidewith 100% identity with the known sequence for human IDUA (GenBankAccession No.: NP_000194).

The pHIR Ab-HC plasmid encodes the heavy chain of a human-mouse chimericHIR Ab, and pHIR Ab-LC encodes the LC of the chimeric HIR Ab. The HC andLC expression vectors are comprised of intronless cDNA open readingframes (orf), and these cDNAs were obtained by RT-PCR of NSO/1 myelomacell lines transfected with chromosomal derived HIR Ab HC and LCintron-bearing vectors as described in detail in Boado et al (2007),Biotechnol Bioeng 96:381-391. See also U.S. patent application Ser. No.11/061,956. The sequence of the HIR Ab HC (SEQ ID NO:7) HIR Ab LC (SEQID NO:8) are shown in FIGS. 1 and 2, respectively. The sequences of theCDRs of the HIR Ab HC (SEQ ID NOs:1-3) and HIR Ab LC (SEQ ID NOs:4-6)are shown in FIG. 3.

The HIR Ab HC and LC intronless cDNA expression vectors are driven bythe cytomegalovirus (CMV) promoter and contain the bovine growth hormone(BGH) polyadenylation (pA) sequence. The engineering of a universal pHIRAb-HC vector was performed by insertion of a single HpaI site at the endof the HIR Ab HC CH3 open reading frame (ORF) by site directedmutagenesis (SDM), as described previously in Boado et al. supra. Allconstructs were validated by bi-directional DNA sequencing.

For the engineering of the expression plasmid encoding the fusionprotein of the heavy chain (HC) of the HIR Ab and IDUA, designatedpCD-HC-IDUA, the human IDUA, minus the 26 amino acid signal peptide, wascloned again by PCR, using the ODNs designated ‘IDUA-signal peptideforward primer’ and ‘IDUA reverse primer.’ in Table 1. The ODNs used forPCR are 5′-phosphorylated for direct insertion of the PCR product intothe HpaI site of the pHIR Ab-HC expression plasmid. The IDUA-signalpeptide forward PCR primer (Table 1) introduces “CA” nucleotides tomaintain the open reading frame and to introduce a Ser-Ser linkerbetween the carboxyl terminus of the CH3 region of the HIR Ab HC and theamino terminus of the IDUA minus the 26 amino acid signal peptide of theenzyme. The IDUA reverse PCR primer introduces a stop codon, “TGA,”immediately after the terminal Pro of the mature human IDUA protein. Thefused IDUA amino acid sequence (SEQ ID NO:9) and the sequence of HIR AbHC-IDUA fusion protein (SEQ ID NO:10) are shown in FIGS. 4 and 5,respectively. A schematic depiction of the bivalent HIR Ab-IDUA fusionantibody is shown in FIG. 6.

DNA sequencing of the expression cassette of the pCD-HC-IDUA encompassed4,369 nt, including a 714 nt CMV promoter, a 9 nt full Kozak site(GCCGCCACC), a 3,276 nt HIR Ab HC-IDUA fusion protein open readingframe, and a 370 nt BGH sequence. The plasmid encoded for a 1,091 aminoacid protein, comprised of a 19 amino acid IgG signal peptide, the 443amino acid HIR Ab HC, a 2 amino acid linker (Ser-Ser), and the 627 aminoacid human IDUA minus the enzyme signal peptide. The predicted molecularweight of the heavy chain fusion protein, minus glycosylation, is118,836 Da, with a predicted isoelectric point (pI) of 8.89.

Example 2 Expression Analysis of IDUA and HIR Ab-IDUA Fusion Protein inCOS Cells

COS cells were plated in 6-well cluster dishes, and were eithertransfected with the pCD-IDUA, or dual transfected with pHIR Ab-LC andpCD-HC-IDUA using Lipofectamine 2000, with a ratio of 1:2.5, μg DNA:uLLipofectamine, and conditioned serum free medium was collected at 3 and7 days. IDUA enzyme activity was measured in both the medium and in theintracellular compartment. The washed monolayer was lysed in 0.4 Msodium formate, pH=3.5, 0.2% Triton X-100; the lysate was sonicated 7sec 3 times on ice, centrifuged, and the supernatant was taken for IDUAenzyme assay (He et al, (1999), Mol Genet Metab, 67:106-112).Transfection of COS cells with pCD-IDUA resulted in high levels of IDUAenzyme activity in both the intracellular compartment and in the mediumat 3 and 7 days following transfection, as shown in Table 2.

TABLE 2 IDUA enzyme activity in COS cells following transfection withpCD-IDUA or cotransfection with pCD-HC-IDUA and pHIR Ab-LC MediumIntracellular activity activity Treatment (nmol/h/mg_(p)) (nmol/h/ml) 3days Lipofectamine 2000  75 ± 7  5.2 ± 1.4 pCD-IDUA 4070 ± 108  1574 ±156 pCD-HC-IDUA, plus  530 ± 34   240 ± 25 pCD-LC-1 7 days Lipofectamine2000  203 ± 80   27 ± 8 pCD-IDUA 7969 ± 858  2286 ± 25 pCD-HC-IDUA, plus1460 ± 136  1194 ± 83 pCD-LC-1 Mean ± SE (n = 3 dishes per point).

Dual transfection of COS cells with the pCD-HC-IDUA and the pHIR Ab-LCresulted in medium levels of IDUA enzyme activity of 240±25 and 1,194±83nmol/hr/mL at 3 and 7 days after transfection, respectively (Table 2).The COS intracellular IDUA enzyme activity at 3 and 7 days is 530±34 and1,460±136 nmol/hr/mg protein, respectively (Table 2). For production oflarger amounts of fusion protein, COS cells were transfected in 10×T500flasks. The 3 day and 7 day medium was pooled, and the 2 L of serum freeconditioned medium was concentrated to 400 mL with tangential flowfiltration (Millipore) followed by purification with protein A affinitychromatography.

The purity of protein A purified fusion protein produced by COS cellswas evaluated with 12% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDSPAGE) with 5% β-mercaptoethanol. Immunoreactivitywas tested with a primary rabbit antibody to human IDUA, provided byProf. E. Neufeld, UCLA, or a primary goat antiserum against human IgGheavy and light chains (Vector Labs, Burlingame, Calif.).

On Western blotting of the purified HIR Ab-IDUA fusion protein, theanti-human IgG antibody reacts with a 130 kDa HC for the fusion protein,and a 50 kDa HC for the chimeric HIR Ab, and the difference in size, 80kDa, is due to the fusion of IDUA (FIG. 9, right panel). The anti-humanIgG antibody reacts equally with the light chain of either the HIRAb-IDUA fusion protein or the HIR Ab, since both proteins are comprisedof the same light chain. The anti-IDUA antibody reacts with the 130 kDaHC of the fusion protein, but not with the HC of the chimeric HIR Ab(FIG. 9, left panel).

Example 3 Analysis of HIR Binding and IDUA Activity

The affinity of the fusion protein for the HIR extracellular domain(ECD) was determined with an ELISA. CHO cells permanently transfectedwith the HIR ECD were grown in serum free media (SFM), and the HIR ECDwas purified with a wheat germ agglutinin affinity column, as previouslydescribed in Coloma et al. (2000) Pharm Res, 17:266-274. The HIR ECD wasplated on Nunc-Maxisoth 96 well dishes and the binding of the HIR Ab, orthe HIR Ab-IDUA fusion protein, to the HIR ECD was detected with abiotinylated goat anti-human IgG (H+L) secondary antibody, followed byavidin and biotinylated peroxidase (Vector Labs, Burlingame, Calif.).The concentration of either HIR Ab or HIR Ab-IDUA fusion protein thatgave 50% maximal binding was determined with a non-linear regressionanalysis.

As shown in FIG. 10 there was comparable binding of either the chimericHIR Ab or the HIR Ab-IDUA fusion protein for the HIR ECD with ED50 of0.61±0.15 nM and 0.93±0.07 nM, respectively.

The IDUA enzyme activity was determined with a fluorometric assay using4-methylumbelliferyl α-L-iduronide (MUBI), which was purchased fromGlycosynth, Ltd. (Cheshire, England). This substrate is hydolyzed to4-methylumbelliferone (4-MU) by IDUA, and the 4-MU is detectedfluorometrically with a Farrand filter fluorometer using an emissionwavelength of 450 nm and an excitation wavelength of 365 nm. A standardcurve was constructed with known amounts of 4-MU (Sigma-Aldrich, St.Louis, Mo.). The assay was performed at 37 C at pH=3.5, and wasterminated by the addition of 1 mL of 0.5 M glycine (pH=10.3). Oneunit=1 nmole substrate/hr (see Kakkis et al., (1994), Prot Expr Purif,5:225-232). The IDUA enzyme activity of the purified HIR Ab-IDUA fusionprotein was 363±37 nmol/hr/ug protein; the assay was linear with respectto both time and mass of fusion protein. Based on these results, weconcluded that the HIR Ab-HC-IDUA fusion/HIR Ab-LC antibody is abifunctional antibody that binds selectively to the IR ECD and retains ahigh level of IDUA activity.

Example 4 HIR Ab-IDUA Fusion Protein Uptake and Biological Activity inMPS Type I Fibroblasts

Type I MPS Hurler fibroblasts and healthy human fibroblasts were grownin 6-well cluster dishes to confluency. The medium was aspirated, wellswashed with phosphate buffered saline (PBS), and incubated with 1 mL ofDulbecco's modified Eagle medium (DMEM) without serum, along with arange of concentrations of the HIR Ab-IDUA fusion protein, for 60 min at37 C. The medium was aspirated, and the wells were washed extensively (1mL/well, 5 washes) with PBS, and the monolayer was taken up in 0.4mL/well of lysis buffer (0.4 M sodium formate, 0.2% Triton X-100,pH=3.5), sonicated on ice 7 seconds 3 times, and microfuged 10 min 4 C.The supernatant was removed for IDUA enzyme activity and bicinchoninicacid (BCA) protein assay. The uptake of the fusion protein was expressedas nmol/hr of IDUA enzyme activity per mg protein.

The HIR Ab-IDUA fusion protein was taken up by MPS Type I fibroblasts(FIG. 11A). The basal IDUA activity in these cells without treatment isvery low (less than 5 nmol/hr/mg of protein). The intracellular IDUAenzyme activity increased in proportion to the concentration of mediumHIR Ab-IDUA. The uptake of the HIR Ab-IDUA by the cells was inhibited55% by the addition of 10 μg/ml murine HIR Ab (p<0.001), but was notinhibited by the addition of 4 mM mannose-6-phosphate (p>0.05). The IDUAenzyme activity in the Hurler fibroblasts approximates 250 nmol/hr/mgpat a medium HIR Ab-IDUA concentration of 2000 ng/mL (FIG. 11A). Thehorizontal line in FIG. 11A denotes the IDUA activity level in healthyhuman fibroblasts.

The effect of the HIR Ab-IDUA fusion protein on cell glycosoaminoglycan(GAG) accumulation was assessed with a 35S incorporation assay (Unger etal, 1994). Type I MPS or healthy human fibroblasts were plated to 6-wellcluster dishes at 250,000 cells/well and grown for 4 days in DMEM with10% fetal bovine serum (FBS). The medium was discarded, the wells werewashed with PBS, and 1 mL/well of low sulfate F12 medium with 10%dialyzed FBS was added, along with 5 mM CaCl2, the HIR Ab-IDUA fusionprotein (0.3 μg/mL), and 10 μCi/mL of 35S-sodium sulfate (Amersham,Chicago, Ill.). Following a 48 hr incubation at 37 C, the medium wasaspirated, the wells were washed with cold PBS (1 mL, 5 washes), and thecells were lysed with 0.4 mL/well of 1 N NaOH. The lysate was heated 60C 60 min to solubilize protein, an aliquot was removed for BCA proteinassay, and the sample was counted for radioactivity with a Perkin ElmerTri-Carb 2100 liquid scintillation counter. The data were expressed as35S CPM per μg protein.

The Hurler fibroblasts, with or without treatment by the HIR Ab-IDUAfusion protein, and the healthy human fibroblasts, were incubated for 48hours in the presence of 35S-sodium sulfate, which is incorporated intointracellular GAGs. Treatment with the HIR Ab-IDUA fusion proteinreduced GAG accumulation in Hurler fibroblasts by 70% as compared tohealthy fibroblasts (p<0.0005) (FIG. 11B).

The prevention of GAG accumulation in Hurler fibroblasts (FIG. 11B)indicated that the HIR Ab-IDUA fusion antibody was directed to thelysosomal compartment of the cell, where GAG accumulates.

To confirm targeting of the HIR Ab-IDUA fusion antibody to the lysosome,confocal microscopy was performed. Type I MPS Hurler fibroblasts weregrown overnight in DMEM with 10% FBS to 50% confluency. The medium wasaspirated, the wells washed well with PBS, and the cells were treatedwith fresh DMEM with no serum and containing 20 μg/mL of the HIR Ab-IDUAfusion protein. Following a 24 hr incubation at 37 C, the medium wasaspirated, the wells washed extensively with cold PBS, and the cellswere fixed with either 100% cold acetone for 20 min at −20 C, or 4%paraformaldehyde at 4 C for 20 min. Following a PBS wash, the plateswere blocked with 10% donkey serum, and then co-labeled with a 1:2000dilution of the rabbit anti-IDUA antiserum, and 10 μg/ml of a mouse MAbto human lysosomal associated membrane protein (LAMP)-1 (BD Pharmingen).Negative control antibodies were the same dilutions of either rabbitserum or mouse IgG. The secondary antibodies (MolecularProbes/Invitrogen) were 5 μg/mL each of 488 conjugated donkey anti-mouseIgG (green channel) and 594 conjugated donkey anti-rabbit IgG (redchannel). The slides were imaged with a Zeiss LSM 5 PASCAL confocalmicroscope with dual argon and helium/neon lasers equipped with ZeissLSM software, as described previously (Zhang et al, (2004), Hum GeneTher, 15:339-350).

The cell IDUA and LAMP-1 immunoreactivity is shown in FIGS. 12A and 12B,respectively. The overlap image in FIG. 12C shows that the fusionprotein was immunoreactive with an anti-IDUA antiserum, and thelysosomal marker, LAMP-1. No immune staining was detected with thecontrol antibodies (FIG. 12D). Based on these results, we concluded thatthe HIR Ab-IDUA fusion antibody was targeted to lysosomes, as would beexpected for IDUA.

Example 5 Analysis of Brain Delivery and Pharmacokinetics of HIR Ab-IDUAFusion Protein in the Rhesus Monkey

The HIR Ab-IDUA fusion protein was iodinated with [125I]-iodine to aspecific activity of 24 μCi/μg, and a trichloroacetic acid (TCA)precipitability of 99%. The fusion protein was iodinated on the same dayas injection into the primate. A 7-year old female Rhesus monkey,weighing 7.2 kg, was obtained from Covance, Inc. (Alice, Tex.), andanesthetized with intramuscular ketamine, and isoflurane by inhalation.The anesthetized primate was administered by a single intravenousinjection a dose of 957 μCi of [125I]-HIR Ab-IDUA fusion protein mixedwith 400 μg (0.06 mg/kg) of unlabeled HIR Ab-IDUA fusion protein in afinal volume of 3 mL. Serum was collected at multiple time points over a120 min period and analyzed for (a) serum 125I radioactivity, and (b)serum IDUA enzyme activity. The serum glucose of the anesthetized,overnight-fasted primate was constant throughout the 120 min studyperiod, and averaged 88±1 mg %, which indicates that the administrationof the HIR Ab fusion protein caused no interference of the endogenousinsulin receptor, and had no effect on glycemic control. At 120 minutesafter drug injection, the animal was euthanized, and brain and organradioactivity was analyzed with a gamma counter; brain was also analyzedwith the capillary depletion method, as described previously (Trigueroet al., (1990), J Neurochem, 54:1882-1888), similar to prior work on thebrain delivery of [¹²⁵I]-labeled murine HIR Ab in the Rhesus monkey(Pardridge et al, (1995), Pharm Res, 12:807-816). The capillarydepletion technique demonstrates the transcytosis of the fusion proteinthrough the BBB in vivo, and into brain.

The delivery of the fusion protein by brain, and other organs, at 120minutes after intravenous injection is expressed as a % of injected dose(ID)/gram organ, and these data are given in Table 3. The fusion proteinis delivered to all parts of brain, as shown by the film autoradiogramof the primate brain at 2 hours after intravenous injection of the[125I]-HIR Ab-IDUA fusion protein (FIG. 12E).

TABLE 3 Brain and organ delivery of an HIR Ab-IDUA Fusion AntibodyTissue % ID/100 g VD (ul/g) Cerebrum gray matter 1.05 ± 0.07  134 ± 8Cerebrum white matter 0.32 ± 0.02  41 ± 2 Cerebellum gray/white 0.74 ±0.17  95 ± 22 Liver 11.7 ± 0.15 1485 ± 18 Spleen 12.1 ± 0.16 1541 ± 20Lung  5.2 ± 0.4  655 ± 48 Kidney  4.0 ± 0.1  505 ± 14 Heart  1.4 ± 0.08 183 ± 9 Skeletal muscle (triceps) 0.48 ± 0.002  61 ± 1 Fat (omental) 1.7 ± 0.4  221 ± 58 Mean ± SD (n = 3 replicates)

The serum ¹²⁵I radioactivity concentration profile, expressed as %injected dose (ID)/mL (FIG. 13A), was fit to a bi-exponential equation,as described previously (Pardridge et al, (1995), Pharm Res,12:807-816), to yield the pharmacokinetic (PK) parameters in Table 4.The parameters for the [¹²⁵I]-fusion protein are compared to the PKparameters reported previously (Coloma et al, (2000), Pharma Res,17:266-274) for the [¹¹¹In]-HIR Ab (Table 4).

The decline in serum radioactivity with time after injection wasparalleled by the decline in serum IDUA enzyme activity (FIG. 13A). Theserum IDUA enzyme activity in the primate before injection of the fusionprotein was 1.5±0.4 units/mL, and was 2120±59, 496±5, 194±20, 67±4,19±1, 12±2, and 14±1 units/mL, at 1, 2.5, 5, 15, 30, 60, and 120 minafter injection, respectively.

The brain delivery of the fusion protein at 2 hours after injection inthe primate is expressed as a volume of distribution (VD), which is theratio of DPM/gram brain divided by the DPM/uL serum (FIG. 13B). Thebrain VD for the fusion protein exceeds 140 μl/gram in the brainhomogenate, and is much larger than the VD for [³H]-mouse IgG2a, whichhas no receptor binding, and does not cross the BBB (Pardridge et al,supra). The brain VD for the [³H]-mouse IgG2a, 18 μl/gram, equals thearterial blood volume of the brain (Ito et al, 2005), which isindicative of lack of transport across the BBB of an antibody that hasno BBB receptor specificity. The brain VD for the [¹²⁵I]-fusion proteinis also ˜140 μl/gram in the post-vascular supernatant (FIG. 13B), asdetermined with the capillary depletion method.

TABLE 4 Pharmacokinetic parameters for [¹²⁵I]-HIR Ab-IDUA fusion proteinand [¹¹¹In]-HIR Ab Parameter [¹²⁵I]-HIR Ab-IDUA [¹¹¹In]-HIR Ab A₁ (%ID/ml)  1.00 ± 0.22 0.15 ± 0.01 A₂ (% ID/ml) 0.077 ± 0.013 0.10 ± 0.01k₁ (min⁻¹)  0.65 ± 0.11 0.12 ± 0.01 k₂ (hr⁻¹)  0.42 ± 0.26 0.11 ± 0.06t_(1/2) ¹ (min)  1.1 ± 0.2  5.8 ± 0.6 t_(1/2) ² (hr)  1.7 ± 1.0  6.3 ±0.6 CL (ml/min/kg)  1.11 ± 0.47 0.22 ± 0.08 Vss (ml/kg)   139 ± 37  116± 11

In table 4A₁, A₂, k₁, and k₂ are the intercepts and slopes of thebi-exponential function describing the decay in plasma concentrationwith time. The parameters for the HIR Ab-IDUA fusion protein weredetermined for the Rhesus monkey in this study, and the parameters forHIR Ab were determined previously in the adult Rhesus monkey (Coloma etal, 2000). All data are normalized for differences in body weight. Thet_(1/2) ¹ and t_(1/2) ² are computed from k₁ and k₂, respectively, andare the half-times of the decay curves for each exponent. Clearance (CL)and steady state volume of distribution (Vss) are computed from A₁, A₂,k₁, and k₂ using previously described pharmacokinetic formulations(Pardridge et al, 1995).

The brain VD of the post-vascular supernatant of the [¹²⁵I]-fusionprotein is equal to the VD of the brain homogenate (FIG. 13B), whichindicated that the fusion protein was transcytosed through the BBB andinto brain parenchyma. The brain VD for the vascular pellet was low,1.1±0.1 μl/g.

Based on these data, we concluded that the HIR Ab-IDUA fusion antibodywas taken up at a high rate into the primate protein, as shown in Table3. This high rate of delivery into the brain was due to the targeting ofthe insulin receptor on the BBB. The fusion protein underwenttranscytosis across the primate BBB in vivo, as demonstrated by thecapillary deletion technique (FIG. 13B).

Importantly, brain delivery of the HIR Ab-IDUA fusion antibody was1.05±0.05% of injected dose per 100 gram brain (Table 3). The size ofthe Rhesus monkey brain is approximately 100 grams; therefore, about 1%of the injected dose is distributed to the primate brain. Owing to thishigh rate of delivery of the fusion antibody into the brain, it will bepossible to produce normal levels of IDUA enzyme activity in the brainof patients with Hurler's syndrome. The delivery of the fusion proteinby brain, expressed as a % of ID/gram, in the human will be reduced, ascompared to the primate, in proportion to body weight. Therefore, theexpected brain delivery of the fusion protein in the human brain isabout 0.1% of the injected dose per 100 gram brain, or about 1% of theID per 1000 g human brain. A normal level of IDUA enzyme activity forthe human brain ranges from 0.5-1.5 units/mg protein (Crow et al,(1983), J Clin Pathol, 36:415-430) and there is a total of about 100,000mg of protein in an average size human brain. Thus, it would be expectedthat delivery to the brain of between 50,000 units to about 150,000units of IDUA activity should suffice to rescue a deficit in brain IDUAactivity as observed in, e.g., Hurler's syndrome. As recombinant IDUAitself does not cross the BBB, this has not been feasible. In contrast,given the observed delivery of the HIR Ab-IDUA fusion antibody into thebrain, and its high IDUA specific activity, we conclude that achievingdelivery of a normalizing level of IDUA activity in the brain ofpatients suffering from an IDUA deficiency (e.g., as in Hurler'ssyndrome) will be achieved by systemic administration of an HIR Ab-IDUAfusion antibody. Further, due to the broad distribution of the fusionantibody in all organs examined (Table 3), systemic administration ofthe HIR Ab-IDUA fusion antibody may also normalize IDUA enzyme activityoutside the CNS in Hurler's patients.

Example 6. Expression Vectors for Permanent Transfection of Host Cell

The genetic engineering of TV-HIRMAb-IDUA was accomplished in severallinear steps, comprised of the following:

-   (1) A “double gene” expression plasmid, designated pCD-HC-IDUA-LC    (FIG. 14) was engineered from 2 precursor plasmids, pCD-HC-IDUA, and    pCD-LC, following linearization of pCD-HC-IDUA by AfeI, and release    of the LC expression cassette with NruI and AfeI, and closure of the    new plasmid with T4 ligase, as shown in FIG. 14.-   (2) A “triple gene” tandem vector (TV) expression plasmid,    designated TV-HIRMAb-IDUA (FIG. 14) was engineered from 2 precursor    plasmids, pCD-HC-IDUA-LC and pwtDHFR, where pwtDHFR encodes for the    wild type (wt) murine dihydrofolate reductase (DHFR). The DHFR    expression cassette was released from pwtDHFR with SmaI and SalI.    The end of SalI was filled with T4 DNA polymerase and    deoxynucleotide triphosphates. In parallel, the pCD-HC-IDUA-LC was    opened with AfeI. The new TV was closed with T4 ligase.

The engineering of the TV was validated by (a) agarose gelelectrophoresis, (b) IDUA expression in COS cells, and (c) bybi-directional DNA sequencing. The entire 7,822 nucleotides (nt) of theTV-HIRMAb-IDUA was subjected to bi-directional DNA sequencing usingcustom oligodeoxynucleotides (ODNs), and the nt sequence is given in SEQID NO. 14. The DNA sequence was comprised of 7,822 nt, which includedthe following domains:

-   -   714 nt cytomegalovirus (CMV) promoter    -   9 nt Kozak sequence (GCCGCCACC)    -   3,276 nt open reading frame (orf) encoding the fusion gene of        the HIRMAb HC and IDUA    -   297 nt bovine growth hormone (BGH) polyA (pA) sequence    -   23 nt linker    -   731 nt CMV promoter    -   9 nt Kozak sequence    -   705 orf encoding the HIRMAb LC    -   291 nt BGH pA    -   254 SV40 promoter    -   9 nt Kozak sequence    -   564 murine DHFR of    -   940 hepatitis B virus (HBV) pA

The TV-HIRMAb-IDUA also included the expression cassette encoding neo,the neomycin resistance gene, to enable selection with G418 (FIG. 14).It was necessary to include the HC fusion gene, the LC gene, and theDHFR gene on a single piece of DNA, or tandem vector (FIG. 14) to allowfor equally high expression of all 3 genes in the transfected host cell.

The TV-HIRMAb-IDUA sequence, from nt 724-3,999 (SEQ ID NO. 14), encodedfor a 1,091 amino acid (AA) HC fusion protein, which was comprised of a19 AA IgG signal peptide, the 442 AA HIRMAb HC, a 3 AA linker, and the627 AA human IDUA enzyme, and is given in SEQ ID. NO. 15. The predictedmolecular weight (MW) of the non-glycosylated HC was 118,795 Daltons(Da) and the predicted isolectric point (pI) of the fusion HC proteinwas 8.85. The TV-HIRMAb-IDUA sequence, from nt 5,060-5,764 (SEQ ID NO.14), encoded for a 234 AA LC protein (SEQ ID NO. 16), which wascomprised of a 20 AA IgG signal peptide, and the 214 AA HIRMAb LC. Thepredicted MW of the LC was 23,398 Da and the predicted pI of the LCprotein was 5.45. The TV-HIRMAb-IDUA sequence, from nt 6,319-6,882 (SEQID NO. 14), encoded for a DHFR protein (SEQ ID NO. 17) that wascomprised of 187 AA.

Example 7. Permanent Transfection of Chinese Hamster Ovary Cells withTV-HIRMAb-IDUA

DG44 Chinese hamster ovary (CHO) cells were grown in serum free HyQSFM4CHO utility medium (HyClone), containing 1×HT supplement(hypoxanthine and thymidine). DG44 CHO cells (5×10⁶ viable cells) wereelectroporated with 5 μg PvuI-linearized TV-HIRMAb-IDUA plasmid DNA. Thecell-DNA suspension is then incubated for 10 min on ice. Cells areelectroporated with BioRad pre-set protocol for CHO cells, i.e. squarewave with pulse of 15 msec and 160 volts. After electroporation, cellsare incubated for 10 min on ice. The cell suspension is transferred to50 ml culture medium and plated at 125 μl per well in 4×96-well plates(10,000 cells per well). A total of 10 electroporations and 4,000 wellsare performed per study.

Following electroporation (EP), the CHO cells are placed in theincubator at 37 C and 8% CO2. Owing to the presence of the neo gene inthe TV, transfected cell lines are initially selected with G418. TheTV-HIRMAb-IDUA also contains the gene for DHFR (FIG. 14), so thetransfected cells are also selected with 20 nM methotrexate (MTX) and HTdeficient medium. Once visible colonies are detected at about 21 daysafter EP, the conditioned medium is sampled for human IgG by ELISA.Wells with high human IgG signals in the ELISA are transferred from the96-well plate to a 24-well plate with 1 mL of HyQ SFM4CHO-Utility. The24-well plates are returned to the incubator at 37 C and 8% CO2. Thefollowing week IgG ELISA is performed on the clones in the 24-wellplates. This is repeated through the 6-well plates to T75 flasks andfinally to 60 mL and 125 mL square plastic bottles on an orbital shaker.At this stage, the final MTX concentration is 80 nM, and the medium IgGconcentration, which is a measure of HIRMAb-IDUA fusion protein in themedium is >10 mg/L at a cell density of 106/mL.

Clones selected for dilutional cloning (DC) are removed from the orbitalshaker in the incubator and transferred to the sterile hood. The cellsare diluted to 500 mL in F-12K medium with 5% dialyzed fetal bovineserum (d-FBS) and Penicillin/Streptomycin, and the final dilution is 8cells per mL, so that 4,000 wells in 40×96-well plates can be plated ata cell density of 1 cell per well (CPW). Once the cell suspension isprepared, within the sterile hood, a 125 uL aliquot is dispensed intoeach well of a 96-well plate using an 8-channel pipettor or a precisionpipettor system. The plates are returned to the incubator at 37 C and 8%CO2. The cells diluted to 1 cell/well cannot survive without serum. Onday 6 or 7, DC plates are removed from the incubator and transferred tothe sterile hood where 125 μl of F-12K medium with 5% dialyzed fetalbovine serum (d-FBS) is added to each well. This selection media nowcontains 5% d-FBS, 30 nM MTX and 0.25 mg/mL Geneticin. On day 21 afterthe initial 1 CPW plating, aliquots from each of the 4,000 wells areremoved for human IgG ELISA, using robotics equipment. DC plates areremoved from the incubator and transferred to the sterile hood, where100 μl of media is removed per well of the 96-well plate and transferredinto a new, sterile sample 96-well plate using an 8-channel pipettor orthe precision pipettor system.

On day 20 after the initial 1 CPW plating, 40×96-well Immunoassay platesare plated with 100 uL of 1 μg/mL solution of Primary antibody, a mouseanti-human IgG in 0.1M NaHCO₃. Plates are incubated overnight in the 4 Crefrigerator. The following day, the ELISA plates are washed with 1×TBST5 times, and 100 uL of 1 ug/mL solution of secondary antibody andblocking buffer are added. Plates are washed with 1× TBST 5 times. 100uL of 1 mg/mL of 4-nitrophenyl phosphatedi(2-amino-2-ethyl-1,3-propanediol) salt in 0.1M glycine buffer areadded to the 96-well immunoassay plates. Plates are read on a microplatereader. The assay produces IgG output data for 4,000 wells/experiment.The highest producing 24-48 wells are selected for further propagation.

The highest producing 24-well plates from the 1 CPW DC are transferredto the sterile hood are gradually subcloned through 6-well dishes, T75flasks, and 125 mL square plastic bottles on an orbital shaker. Duringthis process the serum is reduced to zero, at the final stage ofcentrifugation of the cells and resuspension in SFM.

The above procedures are repeated with a second round of dilutionalcloning, at 0.5 cells/well (CPW). At this stage, approximately 40% ofthe wells show any cell growth, and all wells showing growth alsosecrete human IgG. These results confirm that on average only 1 cell isplated per well with these procedures, and that the CHO cell lineoriginates from a single cell.

Example 8. Manufacturing of CHO-Derived HIRMAb-IDUA Fusion Protein

Following the second round of dilutional cloning, the highest producingcell line secreting the HIRMAb-IDUA fusion protein was propagated inserum free medium to a total volume of 2,000 mL in several 1 L squareplastic bottles on an orbital shaker. The HIRMAb-IDUA fusion protein waspurified from the CHO cell conditioned medium using the followingdown-stream processing:

-   -   Depth filtration with a 0.2 m² 0.65 μm GF filter in series with        an 0.05 m² μm Sartopore-2 μltrafilter    -   Volume reduction to 400 mL using tangential flow filtration        (TFF) system    -   Ultra-filtration with a 0.2 mm μltra-filter and application to a        column of protein A Sepharose 4 Fast Flow. Following application        to the column, the column was eluted with 1 M NaCl, which elutes        DNA non-specifically absorbed to the column, and the product is        eluted as a single peak with 0.1 M sodium acetate/pH=3.7 (FIG.        15A). The acid eluate was neutralized with 1 M Tris base and        concentrated to 5 mL with a Centriprep-30    -   Cation exchange (CATEX) chromatography in bind-elute mode was        performed with a column of SP Sepharose FF equilibrated with        0.02 M MES and 0.05 M NaCl. The conductivity of the sample was        reduced to <5 mS/cm prior to application to the column. The        column was successively eluted with step gradients of 0.02 M        MES/pH=5.5 containing 0.25 M NaCl, 0.35 M NaCl, 0.5 M NaCl, and        1M NaCl. The HIRMAb-IDUA fusion protein eluted in 0.5 M NaCl, as        shown in FIG. 15B.    -   Anion exchange (ANEX) chromatography in flow-through mode was        performed with a column of Q Sepharose FF equilibrated with        0.025 M MES/pH=5.5 and 0.05 M NaCl. The conductivity of the        sample was reduced to <7 mS/cm. The HIRMAb-IDUA fusion protein        eluted in the flow-through as shown in FIG. 15C.

The purity and potency of the CHO derived HIRMAb-IDUA fusion protein wasassessed with the following procedures:

(a) SDS-PAGE.

The CHO-derived HIRMAb-IDUA fusion protein was purified to homogeneitybased on reducing sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), as demonstrated in FIG. 16 (lane 3). Thechimeric HIRMAb is applied to lane 2 of FIG. 16, and HIRMAb-IDUA fusionprotein is applied to lane 3 of FIG. 16. The size of the light chain(LC) of both proteins is the same as both proteins are comprised of thesame LC. The size of the heavy chain (HC) of HIRMAb-IDUA fusion proteinis 130 kDa (lane 3, FIG. 16), whereas the size of the HC of the chimericHIRMAb is 50 kDa (lane 2, FIG. 16), and the difference in size is due tofusion of the 80 kDa IDUA to the HC of the chimeric HIRMAb.

(b) IDUA and Human IgG Western Blot.

The CHO derived HIRMAb-IDUA fusion protein was electrophoresed on a 7.5%SD S-PAGE gel and blotted to nitrocellulose for Western blotting withprimary antibodies to either human IgG (lane 1, FIG. 17), or to humanIDUA (lane 2, FIG. 17). Both the anti-human IgG antibody and theanti-human IDUA antibody reacted specifically with the heavy chain ofHIRMAb-IDUA fusion protein, which migrated with a molecular weight of130 kDa in this reducing gel (FIG. 17).

(c) Human Insulin Receptor (HIR) Binding Assay.

The extracellular domain (ECD) of the HIR was purified by lectinaffinity chromatography from serum free medium conditioned by CHO cellsthat were permanently transfected with the HIR ECD. The HIR ECD wasplated in ELISA wells to bind the chimeric HIRMAb without IDUA fused,and the CHO-derived HIRMAb-IDUA fusion protein. As shown in FIG. 18, theED50 of the chimeric HIRMAb or the HIRMAb-IDUA fusion protein binding tothe HIR is not significantly different, and is 0.75 nM. These dataindicate the affinity of the HIRMAb for the HIR is not affected by thefusion of IDUA to the carboxyl terminus of the IgG. The bindingconstants shown in FIG. 18 were determined by non-linear regressionanalysis of the binding isotherms.

(d) IDUA Enzyme Activity of the HIRMAb-IDUA Fusion Protein.

The IDUA enzyme activity of the CHO-derived HIRMAb-IDUA fusion proteinwas determined with a fluorometric assay using 4-methylumbelliferylα-L-iduronide (MUBI) as the assay substrate. This substrate is hydolyzedto 4-methylumbelliferone (4-MU) by IDUA, and the 4-MU is detectedfluorometrically with a filter fluorometer using an emission wavelengthof 450 nm and an excitation wavelength of 365 nm. A standard curve wasconstructed with known amounts of 4-MU. The assay was performed at 37 Cat pH=3.5, and was terminated by the addition of 1 mL of 0.1 M glycine(pH=10.3). One unit=1 nmol/hr. IDUA enzyme activity of the HIRMAb-IDUAfusion protein was linear with respect to time and concentration (FIG.19). The IDUA enzyme specific activity of the CHO derived theHIRMAb-IDUA fusion protein is 291±9 units per μg protein (FIG. 19). TheIDUA enzyme specific activity of recombinant IDUA is 240 units per μgprotein; therefore, the IDUA enzyme activity of the HIRMAb-IDUA fusionprotein is comparable to that of recombinant IDUA.

(e) Size Exclusion High Performance Liquid Chromatography.

The absence of aggregates in the purified HIRMAb-IDUA fusion protein wasdemonstrated with size exclusion chromatography (SEC) high performanceliquid chromatography (HPLC) using 2 G3000 SWXL columns, 0.78×30 cm, inseries, and an HPLC pump at 0.5 mL/min with detection at 280 nm. Asshown in FIG. 20, the CHO-derived HIRMAb-IDUA fusion protein elutes as asingle peak, removed from the void volume, with no detectableaggregates.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed:
 1. A pharmaceutical composition comprising apharmaceutically acceptable excipient and a therapeutically effectivedose of a fusion antibody having α-L-iduronidase activity, wherein thefusion antibody comprises: (a) a fusion protein comprising an amino acidsequence of an immunoglobulin heavy chain and an α-L-iduronidase (IDUA);and (b) an immunoglobulin light chain; wherein the fusion antibody bindsto an endogenous transferrin receptor of a blood brain barrier (BBB)transport system, wherein the IDUA retains at least 30% of its enzymaticactivity compared to an unfused IDUA, wherein the therapeuticallyeffective dose comprises at least about 1×10⁶ units of α-L-iduronidaseactivity or at least about 140,000 units/Kg of body weight, wherein atleast about 0.5% of the therapeutically effective dose is delivered tothe brain, wherein the amino acid sequence of the IDUA is covalentlylinked to the carboxy terminus of the amino acid sequence of theimmunoglobulin heavy chain, and wherein an IDUA specific activity of thefusion antibody is at least about 250,000 units/mg.
 2. Thepharmaceutical composition of claim 1, wherein the fusion protein of (a)further comprises a linker between the amino acid sequence of the IDUAand the carboxy terminus of the amino acid sequence of theimmunoglobulin heavy chain.
 3. The pharmaceutical composition of claim1, wherein the immunoglobulin heavy chain is an IgG₁.
 4. Thepharmaceutical composition of claim 1, wherein the immunoglobulin lightchain is an immunoglobulin light chain of kappa class.
 5. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is sterile filtered.
 6. The pharmaceutical composition ofclaim 1, wherein the pharmaceutical composition further comprises a saltselected from the group consisting of mineral acid salts and organicacid salts.
 7. The isolated polynucleotide of claim 6, wherein themineral acid salt is selected from the group consisting ofhydrochlorides, hydrobromides, phosphates, and sulfates.
 8. The isolatedpolynucleotide of claim 6, wherein the organic acid salt is selectedfrom the group consisting of acetates, propionates, malonates, andbenzoates.
 9. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition further comprises between 0.01 to 1%polysorbate-80.
 10. The pharmaceutical composition of claim 1, whereinthe pharmaceutical composition further comprises a carbohydrateadditive.
 11. The pharmaceutical composition of claim 10, wherein thecarbohydrate additive is selected from the group consisting of mannitol,sorbitol, and trehalose.